CRISPR Technology Principles and Prospectives

Zagazig University
Faculty of Science
Microbiology and Botany
Department
CRISPR technology; principles

and prospectives
Presented By
Mostafa Ahmed Mahmoud Elkhashab

B.Sc Student, Biotechnology program,
Microbiology and Botany Department
Supervisor
Dr/ Ahmed Abdel-wahed Algazeery

Lecturer of Molecular Genetics, Zoology Department
2019
Acknowledgement
I would like to express my special thanks of gratitude to my

supervisor Dr/ Ahmed Abdel-wahed Algazeery because the
completion of this project could not have been possible without her
expertise.
I would also like to thank my family, friends and colleagues who
believed in me and helped me in finalizing this project.
Dedication
There is nothing in this world that is quite as strong and enduring as a

mother’s love. A mother is often your number one supporter in life, from the
moment you’re born and every day that follows. She nourishes you in every
way she can and will go to the ends of the earth just to see that you are happy
and healthy. A mother offers her invaluable wisdom and advice when you need
it most, even if you don’t realize that you do. I can gratefully say that my
mother who has been all this for me and so much more. That is why I wanted to
take the time and dedicate this piece to her, even though a few loving words are
the least I can do in comparison the person that she is.
Those we love don’t go away, they walk beside us every day unseen,
unheard, but always near still loved, still missed, and very dear. I wish heaven
had visiting hours. Your presence was the best part of my life. Heaven must be
so beautiful...After all, you are there.
To my beloved mom...
CRISPR technology; principles and prospectives
Table of Contents
Table of Contents .............................................................................................. I
List of Abbreviations ...................................................................................... III
Table of figures ................................................................................................ V
Abstract ............................................................................................................. 1
Introduction ....................................................................................................... 2
A Brief History of CRISPR Research from Discovery to Application ............ 4
CONCLUDING REMARKS ........................................................................ 6
The CRISPR-Cas immune system .................................................................... 8
1. CRISPR adaptation .................................................................................. 10
2. Expression of CRISPR RNA and cas genes ............................................ 14
3. Interference .............................................................................................. 15
Diversity and classification of CRISPR-Cas systems .................................... 18
The Class 1 systems ..................................................................................... 19
The Class 2 systems ..................................................................................... 20
Previous Genome editing tools and CRISPR ................................................. 21
The emergence of genome-editing technology ........................................... 21
Zinc finger nucleases ................................................................................... 22
Transcription activator-like effector nucleases ........................................... 25
RNAi ............................................................................................................ 28
CRISPR........................................................................................................ 31
Mechanisms of CRISPR-Cas9 tools......................................................... 33
Advantages of CRISPR-Cas9 gene editing tool ....................................... 38
Delivery systems for CRISPR ........................................................................ 39
Viral Delivery Systems ................................................................................ 39
Non-Viral Delivery ...................................................................................... 40
Applications of CRISPR-Cas9 tools ............................................................... 41
1. Evolution of second-generation CRISPR gene-editing tools .................. 41
I
2. Transcriptional Modulation ..................................................................... 43

3. Rapid Generation of Cellular and Animal Models .................................. 44
4. Epigenetic Control ................................................................................... 44
5. Large-scale genetic and epigenetic CRISPR screenings ......................... 46
6. Agricultural applications ......................................................................... 47
7. Applications in food and industrial biotechnology ................................. 48
8. Biological control applications ................................................................ 49
CRISPR/Cas9 Somatic & Germline editing ................................................... 50
Clinical CRISPR/Cas9 applications on somatic cells ................................. 50
Current clinical trials ................................................................................ 50
CRISPR therapies in development ........................................................... 52
CRISPR/Cas9 germline editing and heritable changes ............................... 53
CRISPR/Cas9 on non-viable human zygotes ........................................... 54
CRISPR/Cas9 on viable human zygotes .................................................. 55
Challenges of CRISPR-Cas9 applications ...................................................... 56
1. Difficulty in generation and delivery of sgRNA array ............................ 56
2. Low efficiency of DNA repair system .................................................... 56
(1) Single-Strand DNA Recombineering (SSDR) ................................... 57
(2) Double-Strand DNA Recombineering (DSDR) ................................. 57
(3) Non-recombineering-based Homologous Recombination (NrHR) .... 58
(4) Non-Homologous End Joining (NHEJ) .............................................. 58
3. Arrangements of CRISPR-Cas system in chromosome or plasmid ........ 59
4. Coupling CRISPR-Cas9 with other genetic tools ................................... 59
5. Off-target effects ...................................................................................... 60
CRISPR and The Ethical dilemma ................................................................. 61
Legislation and logistical considerations ........................................................ 63
References ....................................................................................................... 64
II
List of Abbreviations
Abbreviation Definition
AAV Adeno-associated virus
AdV Adenoviral Vectors
Ago Argonaute protein
AID Activation-induced adenosine deaminase
Cas CRISPR-associated
Cascade CRISPR-associated complex for antiviral
defense
CRISPR Clustered Regularly Interspaced Short
Palindromic Repeats
CRISPRa CRISPR activation
CRISPRdb CRISPRdb database
CRISPRi CRISPR interference
crRNA CRISPR RNA
dCas9 nuclease-deficient Cas9
DSB Double-Stranded Break
dsDNA Double-stranded DNA
dsRNA Double-stranded RNA
epiCas9s epigenetic Cas9 effectors
HDR Homology-directed repair
HNH nuclease domain of Cas9 cleaves the DNA strand
complementary to the RNA guide
Indels Small insertions and deletions
KO Knockout
KRAB Kruppel-associated Box
III
LV Lentivirus
miRNA microRNA
MN Meganuclease
nCas9 Cas9 nickase
ncRNA noncoding RNA
NHEJ Non-Homologous End Joining
PAM Protospacer adjacent motif
piRNA Piwi-interacting RNA
RdRP RNA-dependent RNA polymerase
RNAi RNA interference
RNAP RNA polymerase
RuvC nuclease domain of Cas9 cleaves the DNA strand
not complementary to the RNA guide
sgRNA Single-guide RNA
SID Sin3a Interacting Domain
siRNA short interfering RNA
SpCas9 Streptococcus pyogenes Cas9
SSB Single-Stranded Break
ssDNA Single-stranded DNA
ssRNA Single-stranded RNA
TALE Transcription activator-like effector nuclease
tracrRNA trans-activating crRNA
UGI Uracyl Glycosylase inhibitor protein
WTCas9 Wild-type Cas9
ZFN Zinc-finger nuclease
IV
Table of figures
FIG. 1. TIMELINE OF CRISPR-CAS AND GENOME ENGINEERING RESEARCH FIELDS...................... 6
FIG. 2. THE KEY STEPS OF CRISPR-CAS IMMUNITY ....................................................................... 9
FIG. 3. MODEL OF THE ADAPTATION......................................................................................... 11
FIG. 4. REPAIR OF DOUBLE-STRAND BREAKS ............................................................................. 23
FIG. 5. STRUCTURE OF ZFN........................................................................................................ 24
FIG. 6. STRUCTURE OF TALEN ................................................................................................... 25
FIG. 7. BIOGENESIS OF SIRNA AND MIRNA AND THE MECHANISM GENE SILENCING ................ 29
FIG. 8. STRUCTURE OF CRISPR/CAS9 ......................................................................................... 31
FIG. 9. CRISPR-CAS9-MEDIATED GENOME EDITING................................................................... 32
FIG. 10. CAS9 CATALYTIC DOMAINS .......................................................................................... 33
FIG. 11. CAS9 NUCLEASE (NCAS9) ............................................................................................. 34
FIG. 12. HDR-MEDIATED TARGETED EDITING ............................................................................ 35
FIG. 13. CRISPR ACTIVATION ..................................................................................................... 36
FIG. 14. CRISPR INTERFERENCE ................................................................................................. 37
FIG. 15. SECOND-GENERATION CRISPR GENE EDITING TOOLS .................................................. 41
FIG. 16. TRANSCRIPTIONAL MODULATION ................................................................................ 43
FIG. 17. EPIGENETIC CONTROL .................................................................................................. 45
FIG. 18. IN VIVO AND EX VIVO STRATEGIES FOR CRISPR/CAS9-BASED GENE THERAPIES ........... 51
V
Abstract
In bacteria and archaea, clustered regularly interspaced short palindromic
repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive
immune system against phages and other foreign genetic elements. The diversity,
modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological
revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate
the genomes of cultured cells, animals, and plants, accelerating the pace of
fundamental research and enabling clinical and agricultural breakthroughs. Here
the basic mechanisms that set the CRISPR-Cas toolkit apart from other
programmable gene-editing technologies are described, highlighting the diverse
and naturally evolved systems now functionalized as biotechnologies. CRISPR-
Cas tools at the cutting edge of nucleic acid manipulation that is rewriting
biology. With the emergence of CRISPR technology, targeted editing of a wide
variety of genomes is no longer an abstract hypothetical, but occurs regularly.
1
Introduction
CRISPR and the CRISPR-associated (Cas) protein is a heritable, adaptive
immune system of prokaryotes (bacteria and archaea) that provides them with
memory of previous virus infections and defense against re-infection (R.
Barrangou et al. 2007). Contrary to the human adaptive immune system, CRISPR
is passed onto the next generation of bacteria, rendering the colony immune to
future virus infections (Hille et al. 2018). CRISPR immunity depends on the
integration of the invader’s DNA sequences (virus or plasmid) into the bacterial
genome (Doudna and Charpentier 2014). This allows prokaryotes to detect and
destroy the virus when it returns, by priming the Cas nuclease to target these viral
sequences and cleave them (Doudna and Charpentier 2014). CRISPR stands for
Clustered Regularly Interspaced Short Palindromic Repeats, which are
interrupted by “spacer” sequences (R. Barrangou et al. 2007). These “spacer”
sequences are viral sequences integrated during past viral infections, which when
transcribed into short RNA sequences (CRISPR RNAs), are capable of guiding
the Cas endonuclease to complementary sequences of viral DNA (R. Barrangou
et al. 2007). Upon target identification, Cas binds to the viral DNA and cleaves
it, protecting the prokaryotic cell from infection (R. Barrangou et al. 2007).
CRISPR (clustered regularly interspaced palindromic repeats) was first
described as a gene editing platform in 2012. Since then, this nimble, precise, and
easy-to-use molecular tool has taken the biomedical world by storm‫ــ‬CRISPR was
touted “breakthrough of the year” in 2015 by Science. Why is this particular gene
editing innovation so exciting? In short, CRISPR defies the principle of the “iron
triangle” that for a given product or service “you can have it good, fast, or cheap,
pick two.” CRISPR achieves all three. CRISPR is “good;” unlike previous
methods of genetic engineering, which often resulted in large swaths of DNA
being altered, CRISPR can be used to make precise genetic changes, even at the
single nucleotide level. CRISPR is cheap; unlike its gene editing forerunners,
such as Zinc Finger Nucleases or TALENs, CRISPR is less expensive (150 times
cheaper than TALENs). CRISPR is fast; using just two readily available
molecular components and a straightforward protocol, CRISPR can create
desired gene edits in mere hours. The way CRISPR thus advances genetic
engineering has created immense optimism for its ability to treat heritable
disorders and other diseases with genetic origin (Kraschel and Kofler 2018).
2
The immense intellectual energy surrounding CRISPR is evident in the speed

of its development for clinical application and its uptake by researchers across
the globe. CRISPR based therapeutics are in development to treat numerous
heritable diseases, such as sickle cell disease, cystic fibrosis and hemophilia. The
clinical applications of CRISPR also extend to cancer and infectious diseases.
“The global CRISPR technology market is expected to grow to over $10 billion
by 2027.” (Memi, Ntokou, and Papangeli 2018) Consequentially, CRISPR has
generated both hype and promise in the scientific community as well as the
general public. Prior to 2012, less than 200 scientific articles had been published
on CRISPR. In 2017 alone, nearly 1400 scientific articles referred to CRISPR in
their title or abstract. Today, in labs around the world, CRISPR is used to
understand molecular pathways, create animal models to study disease, and solve
genetic networks (Kraschel and Kofler 2018).
3
A Brief History of CRISPR Research from

Discovery to Application
The initial observation of a repeated 29 nucleotide segment in the genome
of the bacteria Escherichia coli spent years in obscurity before a few
enterprising scientists raised its profile enough to catch the attention of a
larger field of researchers (Ishino et al. 1987). Subsequent advances in both
the basic and applied scientific arenas came rapidly, stimulating more
research.
Although it was not known as CRISPR at the time, the first CRISPR region
was identified in 1987 by Atsuo Nakata and colleagues, who discovered
strange repeats in the iap gene in Escherichia coli (Ishino et al. 1987). They
found five adjacent nucleotide repeats, 29 nucleotides in length, interspaced
with regions 32 nucleotide in length. As more genomic data became available,
similar regions were identified in other bacteria as well as several archaea
(Jansen et al. 2002). Although it was an interesting observation, no function
was assigned to the sequence. Research groups studying these sequences later
agreed on the nomenclature CRISPR, an acronym for clustered regularly
interspaced short palindromic repeats, to describe the sequence composition
of the genomic region (Jansen et al. 2002). In 2002, researchers identified
four CRISPR-associated (cas) genes, which they named cas1- cas4 (Jansen
et al. 2002). A study following up on this observation found additional cas
genes in 27 organisms across two phyla of archaea and ten phyla of bacteria,
expanding the number of known cas genes to 45 (Haft et al. 2005).
A functional relationship for CRISPR/Cas was identified in 2005 by three

groups, who each independently found that CRISPR spacers were derived
from foreign genetic elements (Bolotin et al. 2005; Francisco J M Mojica et
al. 2005; Pourcel, Salvignol, and Vergnaud 2005). The group of Elena Soria
thus hypothesized that CRISPR systems may act to confer resistance to
specific invading DNA elements, such as viruses (Francisco J M Mojica et
al. 2005). To support this hypothesis, mutant bacterial strains were created
with experimentally inserted CRISPR spacers corresponding to specific
bacteriophages; these bacteria gained resistance to only those specific phages
(R. Barrangou et al. 2007).
4
In 2009, it was discovered that CRISPR systems use a protospacer adjacent

motif (PAM) to distinguish between self and nonself (F J M Mojica et al.
2009). The protospacer is the region on the invading DNA corresponding to
the targeting spacer. PAMs are short sequences directly adjacent to the
protospacer on targeted DNA. Different CRISPR systems have different
PAM sequence requirements; the S. pyogenes CRISPR/Cas9 system has an
NGG PAM sequence requirement (F J M Mojica et al. 2009). Another
breakthrough occurred in 2010, when it was discovered that CRISPR systems
function by creating double-stranded breaks in invading phage and plasmid
DNA (Garneau et al. 2010). A major step in the process of understanding a
biological system is the ability to reconstitute it outside of its natural host.
This was made possible by the discovery that the S. thermophilus CRISPR
system only requires one effector protein: Cas9 (Sapranauskas et al. 2011).
Researchers transferred the S. thermophilus CRISPR system, including the
genes cas9, cas1, cas2, csn2, and a specialized CRISPR array onto a plasmid
(Sapranauskas et al. 2011). E. coli transformed with this plasmid were
resistant to genetic material which contained sequences corresponding to the
CRISPR spacers (Sapranauskas et al. 2011). Of the protein-coding genes in
the system, only deletion of the cas9 gene eliminated resistance
(Sapranauskas et al. 2011).
CRISPR development was taken one step further by recreating a functional

CRISPR system in vitro, with only isolated proteins and nucleic acids (Jinek
et al. 2012). A group led by Jennifer Doudna and Emmanuelle Charpentier
found that S. pyogenes Cas9 nuclease and two noncoding RNA species
(crRNA and tracrRNA) were the only required components for targeted
endonuclease activity (Jinek et al. 2012). They also created a chimeric RNA,
which combined the crRNA and tracrRNA, generating a two-component
system for targeted genomic editing (Jinek et al. 2012). The system was
subsequently engineered for human genome editing using an expression
vector containing S. pyogenes cas9 with a codon sequence optimized for
mammalian cell expression and a nuclear localization signal (Cong et al.
2013). This, combined with a chimeric single guide RNA (sgRNA) for
targeting, was used to engineer human and murine cell lines (Cong et al.
2013). This 2013 publication by a group led by Feng Zhang was a milestone
in the CRISPR revolution and set off a rush to explore and adapt the system.
5
Fig. 1. Timeline of CRISPR-Cas and genome engineering research fields.

Key developments in both fields are shown. These two fields merged in 2012
CONCLUDING REMARKS
Only 30 years have passed since one of the authors of this review discovered a
unique repeated sequence in the E. coli genome at the onset of his postdoctoral
career. It was impossible to predict the possible function of this enigmatic
sequence at the time; however, genomic revolution in the mid-1990s, coupled
with the development of powerful bioinformatics tools, eventually enabled
elucidation of the CRISPR functions. CRISPR arrays and Cas proteins, broadly
distributed in the genomes of prokaryotes, especially in archaea, are now known
to constitute the highly efficient acquired immunity system. Although discovery
of the CRISPR-Cas by itself was a great feat of fundamental biology, it also led
to the development of next-generation tools for genetic engineering. The
development of the genome editing technology by CRISPRCas9 reminds of the
times when the PCR was born.
6
When in vitro genetic engineering techniques using restriction endonucleases

and nucleic acid modifying enzymes were established, it was still often a complex
task to clone a single gene (as in the case of the iap gene). However, this difficulty
was alleviated by the invention of PCR using a thermostable DNA polymerase
that profoundly boosted the application of genetic engineering techniques in all
biological laboratories worldwide. The discovery of a thermostable DNA
polymerase was critical for the “PCR revolution,” because it enabled the design
of a PCR apparatus for practical use. Similarly, in the case of genome editing, the
CRISPR revolution was made possible by identifying the right enzymatic system
(Cas9) that could simplify the methodology to exploit the potential of the
CRISPR-Cas system. The curiosity of a mysterious repetitive sequence and a
sustained inquiry mind for elucidating its function brought grand discoveries.
7
The CRISPR-Cas immune system

For each cell on Earth there are about ten viruses (Suttle 2005). Viruses are key
factors in the ecology and evolution of life by acting as predators and facilitators
of genetic exchange. Not surprising, an array of countermeasures can be observed
in their hosts, generally grouped into innate and adaptive immune systems. Innate
(non-specific) systems recognize certain generic features of infection. Restriction
modification and abortive infection are examples of such innate systems in
prokaryotes. Adaptive systems, on the other hand, have the ability to learn to
recognize specific features of pathogens. In humans, B and T cells can learn to
recognize proteins and other structures in order to destroy pathogens and infected
cells. Due to the complexity of that system, the demonstration of an adaptive
immune system in prokaryotes (R. Barrangou et al. 2007) was a surprise. The
prokaryotic system, based on a region of DNA called Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR; (Jansen et al. 2002)), is a
largely standalone system that is capable of functioning in an individual cell, a
necessity for organisms that often display unicellular behavior. The CRISPR-Cas
system targets DNA or RNA as a way of protecting against viruses and other
mobile genetic elements (R. Barrangou et al. 2007; Hale et al. 2009).
The CRISPR locus, first observed in Escherichia coli (Ishino et al. 1987), is
present in about 84% of archaea and 45% of bacteria according to the most recent
update of the CRISPRdb (Grissa, Vergnaud, and Pourcel 2007). The difference
in prevalence could be affected by sampling bias as almost twenty times more
bacteria than archaea have been analyzed. The CRISPR is an array of short
repeated sequences separated by spacers with unique sequences. The CRISPR
can be found on both chromosomal and plasmid DNA. The spacers are often
derived from nucleic acid of viruses and plasmids, an observation that gave rise
to the idea that CRISPRs are part of an anti-virus system (Bolotin et al. 2005;
Francisco J M Mojica et al. 2005; Pourcel, Salvignol, and Vergnaud 2005). By
adding new spacers new viruses can be recognized. The spacers are used as
recognition elements to find matching virus genomes and destroy them.
CRISPR activity requires the presence of a set of CRISPR associated (cas)
genes, usually found adjacent to the CRISPR, that code for proteins essential to
the immune response (R. Barrangou et al. 2007; Brouns et al. 2008). Since the
genome is modified in the process of spacer acquisition, offspring inherit the
protection. New spacers are usually added at one side of the CRISPR, making the
8
CRISPR a chronological record of the viruses the cell and its ancestors have
encountered.
The CRISPR-Cas mediated defense process can be divided into three stages
(Fig. 2). The first stage, adaptation, leads to insertion of new spacers in the
CRISPR locus (Fig. 3). In the second stage, expression, the system gets ready for
action by expressing the cas genes and transcribing the CRISPR into a long
precursor CRISPR RNA (pre-crRNA). The pre-crRNA is subsequently processed
into mature crRNA by Cas proteins and accessory factors. In the third and last
stage, interference, target nucleic acid is recognized and destroyed by the
combined action of crRNA and Cas proteins.
Fig. 2. The key steps of CRISPR-Cas immunity. 1) Adaptation: insertion of new

spacers into the CRISPR locus. 2) Expression: transcription of the CRISPR locus
and processing of CRISPR RNA. 3) Interference: detection and degradation of
mobile genetic elements by CRISPR RNA and Cas protein(s).
9
1. CRISPR adaptation
The adaptation phase provides the genetic memory that is a prerequisite for the
subsequent expression and interference phases that neutralize the re invading
nucleic acids. The insertion of new spacers has been experimentally demonstrated
in several CRISPRCas subtypes; Type I-A (Sulfolobus solfataricus (Erdmann and
Garrett 2012), and Sulfolobus islandicus (Liu et al. 2015)), I-B (Haloarcula
hispanica (M. Li et al. 2014)), I-E (E. coli (Swarts et al. 2012; Yosef, Goren, and
Qimron 2012; Datsenko et al. 2012)) and I-F (Pseudomonas aeruginosa (Cady et
al. 2012) and Pectobacterium atrosepticum (Richter et al. 2014)) and Type II-A
(S. thermophilus (R. Barrangou et al. 2007; Wei, Terns, and Terns 2015; Wei et
al. 2015) and a Streptococcus pyogenes system expressed in Staphylococcus
aureus (Heler et al. 2015)). There are two types of spacer acquisition; naïve, when
the invader has not been previously encountered, and primed, when there is a pre-
existing record of the invader in the CRISPR (Fig. 3) (Fineran and Charpentier
2012).
Although spacer acquisition is observed, the mechanism is only partly
understood. Conceptually, the process can be divided into two steps: protospacer
selection and generation of spacer material followed by integration of the spacer
into the CRISPR array and synthesis of a new repeat. Occasional deletion of
spacers is required to limit the size of the CRISPR, but there is little knowledge
of the mechanism or frequency of such events.
The key factors in spacer integration are Cas1 and Cas2. This function was
suggested early as the proteins are ubiquitous but dispensable for interference
(Brouns et al. 2008). This was later confirmed by overexpression of Cas1 and
Cas2 from a Type I-E system in E. coli, which resulted in spacer integration even
in the absence of all other Cas proteins (Yosef, Goren, and Qimron 2012). Both
Cas1 and Cas2 are nucleases (Babu et al. 2011; Jore et al. 2011; Beloglazova et
al. 2008) and mutations in the active site of Cas1 abolishes spacer integration in
E. coli (Yosef, Goren, and Qimron 2012; Datsenko et al. 2012). Cas1 and Cas2
from E. coli form a complex where one Cas2 dimer binds two Cas1 dimers.
Formation of the complex is required for spacer acquisition but Cas2 nuclease
activity is dispensable. Cas1 preferentially binds CRISPR DNA in a Cas2-
dependent manner, further supporting a direct role in spacer acquisition (Nuñez
et al. 2014). It could be speculated that the Cas1-Cas2 complex both transport the
spacer material and perform spacer integration, which would explain the need for
10
the many Cas1 subunits in the complex. A few additional factors are known to be
required for spacer acquisition: Cas9, Csn2 and tracrRNA in Type II-A (R.
Barrangou et al. 2007; Wei, Terns, and Terns 2015; Heler et al. 2015) and Cas4
in Type I-B (M. Li et al. 2014). The roles of tracrRNA, Csn2 and Cas4 are unclear
but Cas9 probably guides the integration machinery. Host polymerases, ligases
and recombination proteins are likely to perform generic steps in the adaptation,
as such factors can be found in every host cell.
Fig. 3. Model of the adaptation. There are two types of spacer acquisition; naïve, when the
invader has not been previously encountered, and primed, when there is a pre-existing record
of the invader in the CRISPR (Fineran and Charpentier 2012).
11
Spacer selection appears guided by certain sequence elements in the target.

Analysis of target sequences has revealed a short motif next to the target sequence
called protospacer adjacent motif (PAM) that is crucial for discrimination
between self and non-self (F J M Mojica et al. 2009). While initially thought to
be important only for interference, the PAM also has a role in spacer acquisition.
This is supported by the fact that most newly acquired spacers have a PAM next
to their protospacer (Swarts et al. 2012; Datsenko et al. 2012).
In the Type II-A system, Cas9 is responsible for identifying the PAM as
mutations that disable Cas9's PAM recognition result in acquisition from
protospacers without PAM (Heler et al. 2015). In Type I-E, PAM recognition
during spacer acquisition may be different as they are indicated to be identified
by Cas1-Cas2 alone (Yosef, Goren, and Qimron 2012). However, Cascade
increases the frequency of correct PAMs for inserted spacers (Swarts et al. 2012;
Datsenko et al. 2012). In Type I-E, spacers are preferentially incorporated from
extra-chromosomal elements (Datsenko et al. 2012), which is demonstrated to be
a result of a connection between adaptation and replication (Levy et al. 2015). In
Type II-A, spacer acquisition may not be biased toward extra-chromosomal
elements as cells with nuclease-deficient Cas9 demonstrate unbiased spacer
sampling and an increased rate of spacer acquisition in one study (Wei, Terns,
and Terns 2015). Acquisition of self-targeting spacers would not readily be
observed with functional Cas9, as the potential lethality of the events would result
in the cells being lost from the population.
Once located, it is not known if the protospacer is copied or cut out of the
target. The production of spacer material could be linked to other defense
systems, such as the restriction-modification system (Dupuis et al. 2013), similar
to primed spacer acquisition. Such coupling may facilitate the systems'
recognition of “foreign DNA” or provide the system with material suitable for
new spacers. Infection by a phage incapable of reproduction, which e.g. only
packaged a partial viral genome, could act as a vaccine and facilitate adaptation.
How are spacers actually integrated in the CRISPR array? Cas1 nuclease
activity is required for nicking the CRISPR array in E. coli, and Cas1 is possibly
responsible for the integration of the new spacer (Arslan et al. 2014). An in vitro
study with Type I-E Cas1 and Cas2 confirm that the complex can insert DNA
fragments into a CRISPR array by a mechanism reminiscent of retroviral
integrases and transposases (Nuñez et al. 2015). Whether or not the mechanism
12
for spacer integration is conserved between the different systems remains to be

determined.
In both the Type I-E and Type II-A system, it is demonstrated that parts of the
leader and one repeat are required for spacer integration. Further, the leader-
proximal repeat serves as template for synthesis of the new repeat (Yosef, Goren,
and Qimron 2012; Wei et al. 2015), probably by a strand separation mechanism
(Fig. 3). The leader dependence is likely the cause for the observed polar addition
of spacers to the CRISPR (R. Barrangou et al. 2007), although there are reported
exceptions (Erdmann and Garrett 2012). The palindromic nature of many
CRISPR repeats is important to determine the position and direction of spacer
integration into the array (Nuñez et al. 2015). It is indicated that palindromic
repeats form cruciform DNA structures that recruits Cas1 and Cas2 (Fig. 3)
(Nuñez et al. 2015; Arslan et al. 2014), and such structures are known to be a
target for Cas1 cleavage (Babu et al. 2011). Interestingly, in vitro spacer
integration can also be performed at other sequences predicted to form cruciform
structures, in the absence of repeats (Nuñez et al. 2015). Taken together, spacer
integration is directed both by sequence and structure of the CRISPR.
Adaptation has been shown to be coupled to the interference machinery
through primed spacer acquisition, which occurs when there is a targeting spacer
already present in the CRISPR array. The interference machinery and a pre-
existing spacer accelerate the acquisition of subsequent spacers from the same
target. Primed spacer acquisition was first described in the Type I-E system in E.
coli (Datsenko et al. 2012), but has subsequently been reported for I-B in H.
hispanica (M. Li et al. 2014) and I-F in P. atrosepticum (Richter et al. 2014), but
so far not in any Type II or III system. Priming seems to occur by slightly different
processes in the described cases but the exact molecular mechanisms remain
unknown. In Type I-F systems, Cas2 is fused to Cas3 (Makarova, Haft, et al.
2011), further indicating a direct connection between the adaptation and
interference processes. Interestingly, spacers with several mismatches that are
incapable of providing protection against the target still induce primed spacer
acquisition (Fineran et al. 2014). It should be noted that although Cas9 is required
for spacer acquisition in the Type II-A system, this is not an example of primed
spacer acquisition as the requirement is not dependent on a pre-existing spacer
against the target (Heler et al. 2015). The advantages of primed spacer acquisition
are obvious: multiple spacers provide increased resistance against invading DNA,
13
and make it more difficult for target to evolve escape mutants as several sites
would need to be changed simultaneously.
2. Expression of CRISPR RNA and cas genes

The transcription of the CRISPR-Cas loci to generate a RNA protein guide
complex follows a general theme in most organisms but also displays several
type-specific differences. All systems transcribe the CRISPR locus; process the
RNA with Cas ribonucleases and form a CRISPR ribonucleoprotein (crRNP)
complex. In some species, e.g. E. coli, Pyrococcus furiosus and Sulfolobus sp.,
CRISPR transcription initiates in the leader region (Pul et al. 2010; Lillestøl et al.
2009; Hale et al. 2012; Pougach et al. 2010). The leader contains promoter
elements and binding sites for regulatory proteins, in addition to elements
important for spacer integration. A long primary transcript, the pre-crRNA, is
generated and may contain a series of secondary structures (hairpins) if the
CRISPR contains palindromic repeats. The pre-crRNA is processed into smaller
units corresponding to a single spacer flanked by partial repeats. The Cas protein
responsible for the processing, and if that protein is part of a complex or not,
varies with the subtype. Even when the three CRISPR-Cas types naturally co-
exist they do not process each other's pre-crRNA (Carte et al. 2014).
Overall comparison of the three types of CRISPR-Cas systems shows that
Type I and III systems share similarities in pre-crRNA processing as well as in
the structures of the crRNA complexes formed. All Type I and III systems utilize
a Cas6 protein for pre-crRNA processing except Type I-C, which employs Cas5d
(Nam et al. 2012). In Type I-E systems, e.g. in E. coli, the pre-crRNA is cleaved
in a metal-independent mechanism by the Cas6e endoribonuclease (Brouns et al.
2008).
The Cas6e and the crRNA are key components of the E. coli Cascade complex,
which also contains one copy of Cse1, two copies of Cse2, one copy of Cas5e
and six copies of Cas7 (Jore et al. 2011). Cascade has a sea-horse shape where
the Cas7 proteins provide a helical backbone along which the crRNA is displayed
(Wiedenheft, Lander, et al. 2011) held in place by β-hairpin thumbs extending
from Cas7. Cas5e and Cas6e anchor the 5' and 3' ends of the crRNA to opposite
sides of Cascade (Jackson et al. 2014; Mulepati, Héroux, and Bailey 2014; Zhao
et al. 2014). One study has demonstrated that Cascade could be assembled in cells
lacking crRNA and subsequently loaded with crRNA in vitro (Beloglazova et al.
14
2015). The finding suggests that crRNA processing and Cascade assembly do not
need to be an integrated process.
In Type III systems, pre-crRNA maturation also involves a sequence specific
processing step mediated by Cas6 (Hatoum-Aslan, Maniv, and Marraffini 2011;
Marraffini and Sontheimer 2008), followed by a ruler-based sequence-unspecific
crRNA trimming at the 3' end to yield mature crRNAs with a defined 5' end and
variable 3' end (Hatoum-Aslan, Maniv, and Marraffini 2011; J. Zhang et al.
2012). Structural analysis of Type III-A (Csm) and Type III-B (Cmr) complexes
reveal a helical backbone, similar to that of Cascade, against which the crRNA is
aligned. The 5' end of the crRNA is likely anchored by Csm1-Csm4/Cmr2-Cmr3
and the 3' end by Csm5/Cmr1-Cmr6 (Rouillon et al. 2013; Spilman et al. 2013;
Staals et al. 2013).
Type II systems employ a very different mechanism for crRNA biogenesis
where processing is dependent on host RNase III and a trans-encoded small RNA
(tracrRNA) that base pairs with the precrRNA (Deltcheva et al. 2011). In
addition, Type II processing also requires the Cas9 protein (Deltcheva et al. 2011;
Jinek et al. 2012) though its exact role is unclear. Another distinct feature of the
Type II systems is the 5' trimming of the crRNA by an unknown nuclease while
the crRNA tracrRNA remains bound to the Cas9 (Jinek et al. 2012).
3. Interference
The principle of target interference by CRISPR-Cas systems is that crRNA
bound to Cas protein(s) locates the corresponding protospacer to trigger
degradation of the target. The degradation is performed by specific Cas nucleases
(Brouns et al. 2008; Garneau et al. 2010).
In Type I systems Cascade locates the target DNA but the Cas3
nuclease/helicase is needed for interference (Brouns et al. 2008; Makarova, Haft,
et al. 2011; Makarova et al. 2006). Cas3 can be recruited by Cascade upon target
binding or, in the case of Type I-A, be a permanent part of Cascade. In the Type
I-A system the Cas3 nuclease and helicase domains are encoded as separate genes
(Makarova, Haft, et al. 2011). Bioinformatics analysis also suggests a split cas3
gene for I-B systems (Makarova, Haft, et al. 2011; Makarova, Aravind, et al.
2011), but experimental data indicate that this is not always the case (M. Li et al.
2014). However, in all Type I systems the two domains act together to
15
processively degrade the double stranded DNA target (Westra et al. 2012;
Plagens et al. 2014; Maier et al. 2013).
In Type I and II systems interference requires the presence of a PAM sequence
and perfect protospacer-crRNA complementarity in the so-called seed region,
located adjacent to the PAM (Semenova et al. 2011; Wiedenheft, van Duijn, et
al. 2011; Sternberg et al. 2014). The presence of a PAM triggers “non-self
activation”, which prevents the systems from attacking its own CRISPR locus.
In Cascade, the Cas7 thumbs holding the crRNA kink every sixth base out of
position and consequently mismatches at those positions do not affect target
binding (Fineran et al. 2014; Jackson et al. 2014; Mulepati, Héroux, and Bailey
2014; Zhao et al. 2014). A few additional mismatches outside the seed region are
tolerated and do not affect interference (Fineran et al. 2014; Semenova et al.
2011). Cascade can interact non-specifically with DNA (Jore et al. 2011) and
scans for PAMs and seed regions, with the PAM suggested to be detected by Cse1
(Sashital, Wiedenheft, and Doudna 2012). Seed-region base pairing is followed
by pairing the rest of the crRNA, leading to displacement of the non-bound DNA
strand and formation of an R-loop (Jore et al. 2011) and also here Cse1 plays an
important role (Hochstrasser et al. 2014). The binding of the crRNA to the target
causes conformational changes in Cascade and the target DNA (Wiedenheft,
Lander, et al. 2011; Westra et al. 2012) that could be the trigger for Cas3
recruitment. Cas3 then nicks the target DNA and proceeds with progressive
degradation of the target (Westra et al. 2012) while Cascade presumably
dissociates and is ready for action again.
Type II systems require only the Cas9 protein for interference but unlike Type
I and III systems it needs not just crRNA, but also a tracrRNA bound to Cas9 and
the crRNA to perform target recognition and degradation (Deltcheva et al. 2011).
Cas9 structures from S. pyogenes and Actinomyces naeslundii reveal separate
lobes for target recognition and nuclease activity, accommodating the crRNA-
DNA heteroduplex in a positively charged groove at their interface (Nishimasu
et al. 2014; Jinek et al. 2014). The recognition lobe is important for binding
crRNA and target DNA, and the nuclease lobe contains the HNH and RuvC
nuclease domains that cleave the complementary and non-complementary
strands of the target, respectively (Nishimasu et al. 2014). As the crRNA-induced
reorientation of structural lobes facilitates DNA substrate binding, loading of
crRNA is proposed as a key step in Cas9 activation (Jinek et al. 2014).
16
The Csm complex in Type III-A typically includes six different proteins but
the nuclease is not yet identified (Rouillon et al. 2013). The Cmr complex in III-
B includes six or seven different proteins (Rouillon et al. 2013; Spilman et al.
2013; J. Zhang et al. 2012) and the Cmr4 protein cleaves the target (Zhu and Ye
2015; Ramia et al. 2014; Benda et al. 2014). Early findings indicated that Csm
complexes target DNA (Marraffini and Sontheimer 2008) and Cmr complexes
targets RNA (Hale et al. 2009; Staals et al. 2013; Zebec et al. 2014), but a more
complex picture is now emerging. In Thermus thermophilus and S. thermophilus
the Csm complex targets RNA, and in T. thermophilus, which harbors both Type
III-A and III-B systems, the Csm and Cmr complexes share crRNA (Staals et al.
2014; Tamulaitis et al. 2014). Targeting of RNA and DNA by the same Cmr
complex has been demonstrated in S. islandicus (Deng et al. 2013; W. Peng et al.
2015). No PAMs are detected for Type III systems, instead the discrimination
between self and non-self is achieved by an extension of crRNA base pairing into
the repeat region of host DNA, which results in “self inactivation”, a
fundamentally different process to the PAM recognition used by Type I and II
systems (Marraffini and Sontheimer 2010).
An additional aspect of interference has been demonstrated for a Type III-A
system in S. aureus, where the system is prevented from attacking un transcribed
targets such as lysogenized phages (Goldberg et al. 2014). Such a system makes
sense since it prevents potential degradation of the host's own chromosome. The
mechanism behind the system is not known and it remains to be determined if
this is a widespread feature of CRISPR-Cas systems.
17
Diversity and classification of CRISPR-Cas

systems
The bacterial and archaeal CRISPR-Cas systems of adaptive immunity show
remarkable diversity of protein composition, effector complex structure, genome
locus architecture and mechanisms of adaptation, pre-CRISPR (cr)RNA
processing and interference. The CRISPR-Cas systems belong to two classes,
with multi-subunit effector complexes in Class 1 and single-protein effector
modules in Class 2. The newly characterized type VI systems are the first among
the CRISPR-Cas variants to exclusively target RNA. Unexpectedly, in some of
the class 2 systems, the effector protein is additionally responsible for the pre-
crRNA processing. Comparative analysis of the effector complexes indicates that
Class 2 systems evolved from mobile genetic elements on multiple, independent
occasions.
CRISPR- Cas is a programmable form of immunity that can adapt to target any
sequence and therefore is not under pressure to evolve an immense diversity of
specificities as is the case for restriction-modification enzymes, the most
abundant form of innate immunity in prokaryotes (Pingoud, Wilson, and Wende
2014). Nevertheless, like any defense system, CRISPR-Cas is engaged in an
incessant arms race with viruses, which results in rapid evolution of cas genes
(Takeuchi et al. 2012)and notable diversity of the gene repertoires and
architectures of the CRISPR-cas loci translating into diversification of the actual
defense mechanisms (Makarova, Haft, et al. 2011; Makarova et al. 2015). More
specifically, the diversification of the CRISPR Cas systems is likely to be partly
driven by their competitive coevolution with virus-encoded dedicated anti-
CRISPR proteins (Joe Bondy-Denomy et al. 2012; Joseph Bondy-Denomy et al.
2015; Pawluk et al. 2016).
Despite this extensive diversification, comprehensive comparative analysis has
revealed major unifying themes in the evolution of CRISPR-Cas. These common
trends include multiple contributions of mobile genetic elements to the
emergence of the CRISPR-Cas immunity and its distinct variants; extensive
duplication of cas genes yielding functionally versatile effector complexes; and
modular organization, with extensive recombination of the modules (Mohanraju
et al. 2016; Makarova et al. 2015; Makarova, Wolf, and Koonin 2013). The two
18
principal modules of the CRISPR-Cas systems consist of the suites of genes

encoding proteins involved in adaptation (spacer acquisition) and effector
functions, that is, pre-crRNA processing, and target recognition and cleavage.
The adaptation module is largely uniform across the diversity of the CRISPR-Cas
systems and consists of the endonuclease Cas1 and the structural subunit Cas2
(Amitai and Sorek 2016). In contrast, the effector modules are highly variable
between CRISPR- Cas types and subtypes. Various proteins involved in ancillary
roles, such as regulation of the CRISPR response and other, still poorly
characterized functions, can be assigned to a third, accessory module (Mohanraju
et al. 2016; Makarova et al. 2015; Makarova, Wolf, and Koonin 2013).
The fast evolution and variability of the CRISPR-Cas systems makes their
classification a daunting task. Given the absence of universal cas genes and the
frequent modular recombination, a single classification criterion is impractical.
Therefore, a multipronged approach to CRISPR-Cas classification has been
adopted that takes into account the signature cas genes that are specific for
individual types and subtypes of CRISPR-Cas, sequence similarity between
multiple shared Cas proteins, the phylogeny of Cas1 (the best conserved Cas
protein), the organization of the gene in the CRISPR-cas loci and the structure of
the CRISPR themselves (Makarova et al. 2015; Makarova, Aravind, et al. 2011).
The combined application of these criteria resulted in the currently adopted
classification scheme that partitions the CRISPR-Cas systems into two distinct
classes characterized by different principles of the effector module design.
The Class 1 systems

The Class 1 systems include the most common and diversified type I, type III
that is represented in numerous archaea but is less frequent in bacteria, as well as
the rare type IV that includes rudimentary CRISPR-cas loci lacking the adaptation
module. The effector complexes of type I and type III CRISPR-Cas display
elaborate architectures, with a backbone consisting of paralogous RAMPs
(Repeat-Associated Mysterious Proteins), such as Cas7 and Cas5, containing the
RRM (RNA Recognition Motif) fold and additional ‘large’ and ‘small’ subunits
(Zhao et al. 2014; van der Oost et al. 2014; Jackson et al. 2014; Jackson and
Wiedenheft 2015; Hochstrasser et al. 2014, 2016, Staals et al. 2013, 2014). These
effector complexes contain one Cas5 subunit and several Cas7 subunits. The
complex accommodates the guide RNA that consists of the spacer and a portion
19
of a repeat. The Cas5 subunit binds the 50 handle of the crRNA and interacts with
the large subunit (Cas8 in type I and Cas10 in type III). The small subunit is
typically present in several copies and interacts with the crRNA backbone bound
to Cas7. Notably, the length of the bound spacer correlates with the number of
Cas7 subunits in the backbone of the complex (Hatoum-Aslan et al. 2013;
Kuznedelov et al. 2016; Luo et al. 2016). Although the sequences of the protein
sub-units in type I and type III effector complexes show little sequence similarity
to each other, the presence of the homologous RAMPs in both types of complexes
and the overall structural similarity revealed by cryo-electron microscopy leave
little doubt in the common origin of these complexes (Makarova et al. 2015;
Makarova, Aravind, et al. 2011; Jackson and Wiedenheft 2015). An additional
RAMP, Cas6, is loosely associated with the effector complex and typically
functions as the repeat-specific RNase in the pre-crRNA processing (Charpentier
et al. 2015; O. Niewoehner and Jinek 2016).
The Class 2 systems

The effector modules of Class 2 consist of a single, large, multidomain protein
each, such that the respective CRISPR-cas loci have a much simpler and more
uniform organization than those of Class 1. At the time of the construction of the
current formal CRISPR-Cas classification (Makarova et al. 2015), Class 2
included 3 subtypes of the thoroughly characterized type II, with the effector
endonuclease Cas9, the widely used genome editing enzyme, and type V, with
the predicted effector protein Cpf1. Type V was included into the classification
scheme for the first time and remained experimentally uncharacterized. The
subsequent demonstration that Cpf1 was indeed an active RNA-guided
endonuclease that, unlike Cas9, did not require the additional trans-activating
CRISPR (tracr)RNA for target cleavage (Zetsche et al. 2015), raised interest in
comprehensive characterization of the diversity of the Class 2 CRISPR-Cas
systems by genome and metagenome mining.
20
Previous Genome editing tools and CRISPR

The emergence of genome-editing technology
The classical method for gene modification is homologous recombination.
This approach has been widely used in mouse embryonic stem cells to generate
germline knockout or knock in mice (Smithies et al. 1985; Kirk R Thomas and
Capecchi 1987). A disadvantage is that it typically takes more than a year to
generate a genetically modified mouse using the standard approach. Furthermore,
similar attempts at using homologous recombination in human cells have proven
to be far more challenging, and alternative approaches to knock down gene
expression, such as antisense oligonucleotides and short interfering RNAs, have
instead become standard. However, these approaches only transiently reduce
gene expression, and the effect is usually incomplete and can often affect off
target genes (Qiu, Adema, and Lane 2005; L. Wang et al. 2014). These
shortcomings have fueled the demand for more effective methods of gene
modification. A new wave of technology that is variously termed “gene editing,”
“genome editing,” or “genome engineering” has emerged to address this demand
by giving investigators the ability to precisely and efficiently introduce a variety
of genetic alterations into mammalian cells, ranging from knock in of single
nucleotide variants to insertion of genes to deletion of chromosomal regions.
21
Zinc finger nucleases

Zinc finger nucleases (ZFNs) are increasingly being used in academia and
industrial research for a variety of purposes ranging from the generation of animal
models to human therapies (Urnov et al. 2010). ZFNs are fusion proteins
comprising an array of site-specific DNA-binding domains — adapted from zinc
finger–containing transcription factors — attached to the endonuclease domain
of the bacterial FokI restriction enzyme. Each zinc finger domain recognizes a 3-
to 4-bp DNA sequence, and tandem domains can potentially bind to an extended
nucleotide sequence (typically with a length that is a multiple of 3, usually 9 bp
to 18 bp) that is unique within a cell’s genome.
To cleave a specific site in the genome, ZFNs are designed as a pair that
recognizes two sequences flanking the site, one on the forward strand and the
other on the reverse strand. Upon binding of the ZFNs on either side of the site,
the pair of FokI domains dimerize and cleave the DNA at the site, generating a
double-strand break (DSB) with 5' overhangs (Urnov et al. 2010). Cells repair
DSBs using either (a) nonhomologous end joining (NHEJ), which can occur
during any phase of the cell cycle, but occasionally results in erroneous repair, or
(b) homology-directed repair (HDR), which typically occurs during late S phase
or G2 phase when a sister chromatid is available to serve as a repair template (Fig.
4).
The error-prone nature of NHEJ can be exploited to introduce frameshifts into
the coding sequence of a gene, potentially knocking out the gene by a
combination of two mechanisms: premature truncation of the protein and
nonsense-mediated decay of the mRNA transcript, the latter of which is not
always particularly efficient (Fig. 4). Alternatively, HDR can be utilized to insert
a specific mutation, with the introduction of a repair template containing the
desired mutation flanked by homology arms (Fig. 4). In response to a DSB in
DNA, HDR utilizes another closely matching DNA sequence to repair the break.
Mechanistically, HDR can proceed in the same fashion as traditional homologous
recombination, using an exogenous double-stranded DNA vector as a repair
template (Rouet, Smih, and Jasin 1994). It can also use an exogenous single-
stranded DNA oligonucleotide (ssODN) as a repair template. For ssODNs,
homology arms of as little as 20 bp can enable introduction of mutations into the
genome (Radecke et al. 2010; Soldner et al. 2011; Chen et al. 2011). In either
case, the efficiency can be sufficiently high such that antibiotic selection to
22
identify correctly targeted clones is unnecessary (Soldner et al. 2011; Urnov et al.
2005).
Fig. 4. Repair of double-strand breaks. DNA double-strand breaks (DSBs) are typically
repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR).
Despite the advantages of genome editing with ZFNs, there are several
potential disadvantages. It has not proven to be straightforward to assemble zinc
finger domains to bind an extended stretch of nucleotides with high affinity
(Ramirez et al. 2008). This has made it difficult for nonspecialists to routinely
engineer ZFNs. To surmount this difficulty, an academic consortium has
developed an open-source library of zinc finger components and protocols to
perform screens to identify ZFNs that bind with high affinity to a desired
sequence (Maeder et al. 2008, 2009); nonetheless, it can still take months for
nonspecialists to obtain optimized ZFNs. Another potential disadvantage is that
target site selection is limited — the open-source ZFN components can only be
used to target binding sites every 200 bps in random DNA sequence. While this
may be a nonissue if an investigator seeks to knock out a gene, since a frameshift
introduced anywhere in the early coding sequence of the gene can produce the
desired result, it may present challenges if a particular site is required, e.g., to
knock in a specific mutation. Since the introduction of the open-source platform,
alternative platforms to engineer optimized ZFNs have emerged, each with
varying degrees of speed, flexibility in site selection, and success rates (Sander et
al. 2010; Gupta et al. 2012; Bhakta et al. 2013; Gaj et al. 2012; H. J. Kim et al.
2009).
23
Finally, a significant concern about the use of proteins designed to introduce

DSBs into the genome is that they will do so not only at the desired site but also
at off-target sites. In one study in which ZFNs were used for genome editing in
human pluripotent stem cells, the investigators identified ten possible off-target
genomic sites based on high-sequence similarity to the on-target site and found a
single off-target mutation in the 184 clones assessed (Hockemeyer et al. 2009).
Two subsequent studies of ZFNs seeking to identify potential off-target sites for
several ZFN pairs revealed off-target events at numerous loci in a cultured human
tumor cell line (Gabriel et al. 2011; Pattanayak et al. 2011). Thus, investigators
should be cognizant of the possibility that ZFNs designed for a particular purpose
may incur undesired off-target events at a low rate. One strategy to reduce off-
target events is to use a pair of ZFNs that have distinct FokI domains that are
obligate heterodimers (Doyon et al. 2010; Szczepek et al. 2007; Miller et al.
2007). This prevents a single ZFN from binding to two adjacent off-target sites
and generating a DSB; rather, the only way an off-target event could occur is if
both ZFNs in a pair bind adjacently and thus allow the FokI dimer to form.
Another strategy that has been demonstrated to reduce off-target events is the
introduction of purified ZFN proteins into cells (Gaj et al. 2012).
Fig. 5. Structure of ZFN. ZFN dimer bound to its target DNA, each one has zinc-finger protein
(ZFP) at the N-terminus and FokI nuclease domain C-terminus.
24
Transcription activator-like effector nucleases

The recent discovery of a class of proteins called transcription activator-like
effectors (TALEs), exclusive to a group of plant pathogens, has led to the
characterization of a novel DNA-binding domain, termed TALE repeats. The
naturally occurring TALE repeats comprise tandem arrays with 10 to 30 repeats
that bind and recognize extended DNA sequences (Bogdanove and Voytas 2011).
Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids
(termed the repeat-variable di-residue [RVD]) conferring specificity for one of
the four DNA base pairs (Moscou and Bogdanove 2009; Boch et al. 2009;
Morbitzer et al. 2010; Streubel et al. 2012; Cong et al. 2012). Thus, there is a one-
to-one correspondence between the repeats and the base pairs in the target DNA
sequences. Elucidation of the RVD code has made it possible to create a new type
of engineered site-specific nuclease that fuses a domain of TALE repeats to the
FokI endonuclease domain, termed TAL effector nucleases (TALENs) (refs.
(Christian et al. 2010; Miller et al. 2010; T. Li et al. 2011) and Fig. 6). TALENs
are similar to ZFNs in that they can generate DSBs at a desired target site in the
genome and so can be used to knock out genes or knock in mutations in the same
way (Fig. 4).
Fig. 6. Structure of TALEN. Pair of TALENs targets the DNA; each construct has TALE
protein domain at the amino acid terminus and FokI at carboxyl terminus. The left TALE and
right TALE repeat domains recognized 30–40 bp of the target DNA; each repeat has 33–35
amino acids that recognize one single base via its repeated variable diresidues (RVDs).
25
In comparison with ZFNs, TALENs have turned out to be much easier to

design. The RVD code has been employed to engineer many TALE repeat arrays
that bind with high affinity to desired genomic DNA sequences; it appears that
de novo–engineered TALE repeat arrays will bind to desired DNA sequences
with high affinity at rates as high as 96% (Miller et al. 2010; Hockemeyer et al.
2011; Reyon et al. 2012). TALENs can be designed and constructed in as short a
time as two days and in as large a number as hundreds at a time (Reyon et al.
2012; Cermak et al. 2011); indeed, a library with TALENs targeting all of the
genes in the genome has been constructed (Y. Kim et al. 2013).
One potential advantage over ZFNs is that the TALE repeat array can be easily
extended to whatever length is desired. Whereas engineered ZFNs typically bind
9- to 18-bp sequences, TALENs are often built to bind 18-bp sequences or even
longer, though recent evidence suggests that the use of larger TALENs may result
in less specificity (Guilinger et al. 2014). Another advantage of TALENs over
ZFNs is that there appear to be fewer constraints on site selection; in theory, there
are multiple possible TALEN pairs available for each bp of a random DNA
sequence (Reyon et al. 2012).
As with ZFNs, off-target effects are a significant concern with TALENs. A
study in which TALENs were used for genome editing in human pluripotent stem
cells found low but measurable rates of mutagenesis at some of 19 possible off-
target sites based on sequence similarity to the on-target sites (Hockemeyer et al.
2011). Although comparative data are scarce, one study found that for TALENs
and ZFNs targeting the same site in the CCR5 gene, the TALENs produced fewer
offtarget mutations than the ZFNs at a highly similar site in the CCR2 gene
(Mussolino et al. 2011). Furthermore, the ZFNs produced greater cell toxicity
(i.e., inhibited their growth) when introduced into cells compared with the
TALENs (however, it should be noted that ZFN versus TALEN protein
concentrations were not normalized, and the ZFNs used in this study were of a
design that was several years old, rather than being state-of-the-art ZFNs). As
with ZFNs, TALENs with obligate heterodimer FokI domains are routinely used
to minimize the possibility of off-target events. Recently, whole-genome
sequencing studies of human pluripotent stem cell clones edited with TALENs
showed that the overall off-target event rate is very low (Smith et al. 2014; Suzuki
et al. 2014).
26
A clear disadvantage of TALENs is their significantly larger size compared

with ZFNs. The typical size for a cDNA encoding a TALEN is approximately 3
kb, whereas a cDNA encoding a ZFN is only approximately 1 kb. In principle,
this makes it harder to deliver and express a pair of TALENs into cells compared
with ZFNs, and the size of the TALENs makes them less attractive for therapeutic
applications in which they must be delivered in viral vectors with limited cargo
size (such as adenoassociated virus [AAV], with less than 5 kb) or as RNA
molecules. Furthermore, the highly repetitive nature of the TALENs may impair
their ability to be packaged and delivered by some viral vectors (Holkers et al.
2013), though this can apparently be overcome by diversifying the coding
sequences of the TALE repeats (Yang et al. 2013).
27
RNAi
RNA interference (RNAi) is the common gene silencing mechanism in
eukaryotes, which 20–30 nt of small noncoding RNA (ncRNA) act as regulators
for RNA stability, host defense, transcription, and translation (Carthew and
Sontheimer 2009; Ding and Voinnet 2007). Active RNAi requires an
Argonaute/P-element induced wimpy (Piwi) protein as the core component of
RNA-induced silencing complex (RISC) (Höck and Meister 2008), guiding
ncRNA to the target gene (Carmell et al. 2002; Cerutti and Casas-Mollano 2006).
The family of Argonaute proteins has two subfamilies: Ago (Argonaute) that is
ubiquitously expressed in eukaryotes and Piwi that is expressed in germ lines in
humans (Carmell et al. 2002; Hutvagner and Simard 2008). Argonaute proteins
are the key players in gene-silencing mechanisms guided by small ncRNA. There
are three categories of RNAi in eukaryotes, namely, short interfering RNAs
(siRNAs), microRNAs (miRNAs), and Piwi-interacting RNA (piRNA) (Höck
and Meister 2008). Biosyntheses of siRNA and miRNA are derived from double-
stranded RNA (dsRNA) by action of Dicer, generating short duplex (21–25 nt).
siRNAs are derived from exogenous dsRNA, endogenous transcript, from long
hairpin transcripts (Ghildiyal and Zamore 2009), that require RNA-dependent
RNA polymerase (RdRP) to generate dsRNA from single-stranded RNA
(ssRNA) (Dang et al. 2011; Gent et al. 2010), that completely matched to mRNA
target. While, miRNAs are generated from miRNA-encoding genes that generate
ssRNA that form hairpin structure. RNAi has three components: Dicer,
Argonaute, and RdRP proteins identified in eukaryotes, ensuring the involvement
of RNAi as common regulatory mechanism in all eukaryotes (Dang et al. 2011).
In RNAi biogenesis, the aberrant RNA is recognized and converted into dsRNA
by RdRP, subsequently processed by Dicers into siRNA, then loaded to
Argonaute proteins (Nakayashiki et al. 2005). In filamentous fungi, RNAi is the
common defense mechanism against viral and transposon invasion (Dang et al.
2011) (Fig. 7). The expression of dsRNA in N. crassa triggers a robust dsRNA-
activated transcriptional program that subsequently activates the expression of all
RNAi genes including Ago proteins, Dicer as antiviral mechanisms (Dang et al.
2011). In asexual stages of N. crassa, RNAi silencing phenomena “quelling”
occurred by introduction of repetitive DNA sequences triggering the
posttranscriptional gene silencing (Fulci and Macino 2007). Quelling is the most
common gene silencing mechanism in Ascomycetes at the post-transcriptional
level, in which the generated siRNA targets the mRNA to be silenced (Fulci and
28
Macino 2007); it is controlled by qde-1 (encodes RdRP), qde-2, qde-3, and Dicer
proteins (Catalanotto et al. 2000). However, in sexual stage, there are two
silencing mechanisms, namely, repeated-induced point (RIP) mutations and
meiotic silencing by unpaired DNA (Selker and Stevens 1987; Shiu et al. 2001).
Fig. 7. Biogenesis of siRNA and miRNA and the mechanism gene silencing. Different
sources of RNA can be adopted as dsRNA by RDRP. The dsRNA can be processed by Dicer
to siRNA. siRNA are 20–30 nt that loaded into the Argonaute proteins; the guide strand of
siRNA is further assembled into the RISC (siRISC), while the passenger strand is degraded.
The guide strand on siRISC targets the RNA by Watson-Crick base pairing, thus causing
silencing to the target genes. The miRNAs are processed from existing precursor Pri-miRNA,
which is processed by Drosha into pre-miRNA in nucleus and exported to cytoplasm and
further processed by Dicer to form miRNA, which is subsequently assembled with the
Argonaute proteins to form mature RISC for recognizing and targeting RNA by Watson-Crick
base pairing, silencing the target genes (Carthew and Sontheimer 2009).
miRNA is a common regulator to the endogenous genes, while siRNA is the

defender of genome integrity against invasive nucleic acids of virus and
transposons (Carthew and Sontheimer 2009). Single strand of siRNA (siRISC)
and miRNA (miRISC) are associated with the RISCs (Carthew and Sontheimer
2009; S. M. Hammond et al. 2000); for both types, the target gene to be silenced
are specified by RNA Watson-Crick base pairing. The main differences between
miRNA and siRNA are the following: miRNAs are endogenous from the own
cell genome, processed from stem-loop precursors with incomplete dsRNA,
while siRNAs mainly originated exogenously from virus, transposons, and
29
excised from long, fully complementary dsRNA (Tomari and Zamore 2005). The
biogeneses of siRNA and miRNA are illustrated in (Fig. 7).
The siRNA guide strand directs the RISC for perfect pairing with target RNA
that subsequently degraded. RNA degradation is induced by Piwi domain of Ago
protein, “Slicer” activity cleaving the phosphodiester bond between the target
nucleotide that base paired to siRNA residues 10 and 11 (counting from the 5'
end) generating product with 5'- monophosphate and 3'-hydroxyl termini
(Carthew and Sontheimer 2009; Tomari and Zamore 2005) Once doing this initial
cut, the cellular exonuclease cleaves the fragment to complete the degradation
process (ORBAN and IZAURRALDE 2005) and the RISC cleavage product is
further used for oligouridylation, triggering the exonucleolytic activity (Shen and
Goodman 2004). The cleavage products were released, and the RISC becomes
free for additional targets (Carthew and Sontheimer 2009).
Dicer (endoribonuclease Dicer, helicase with RNase motif) is a member of
RNase III family that cleaves the dsRNA and pre-miRNA into short dsRNA
fragments siRNA and miRNA, respectively (Agrawal et al. 2003). RNase III
members are few nucleases that precisely cleave the dsRNA into 3' overhang of
2–3 nt of hydroxyl termini and 5'phosphate (Agrawal et al. 2003). This enzyme
was firstly identified in Drosophila extract that has the ability to produce
fragments of about 22 nt, initiating the RNAi processing (Bernstein et al. 2001).
Dicer has four domains: amino terminal helicase domain, dual RNase III motif,
dsRNA binding domain, and PAZ domain (Piwi, Argonaute, Zwille domain)
which is shared with RDE1/QDE2/Argonaute family (Catalanotto et al. 2000;
Tabara et al. 1999). Argonaute proteins are the core of RNA silencing system,
Ago clade, associated with miRNA and siRNA (Meister and Tuschl 2004; Tomari
and Zamore 2005).
30
CRISPR
Researchers have adapted the bacterial CRISPR immune system to create a
gene editing tool that can make targeted changes to the DNA of any organism.
The most common adaptation is CRISPR/Cas9, which consists of the Cas9
endonuclease and a short noncoding guide RNA (gRNA) that has two
components: a target-specific CRISPR RNA (crRNA) and a helper trans-
activating RNA (tracrRNA) (Brouns et al. 2008; Karvelis et al. 2013). The gRNA
unit guides Cas9 to a specific genomic locus via base pairing between the crRNA
sequence and the target sequence (Pattanayak et al. 2013). The target DNA
sequence, known as the “protospacer”, must be both complementary to the gRNA
but also contain a three-nucleotide Protospacer-Adjacent Motif (PAM), on the 5'
end of the target strand (Marraffini and Sontheimer 2010; O’Connell et al. 2014;
Anders et al. 2014) (Fig. 8). The PAM sequence is required for Cas enzyme
compatibility and provides a mechanism for the system to discriminate between
bacterial and invader sequence (Marraffini and Sontheimer 2010; O’Connell et
al. 2014). Single gRNAs, constructed through fusion of guide sequence-
containing crRNA to tracrRNA have been shown to work as efficiently as the
individual elements, further simplifying the use of the CRISPR/Cas9 system
(Jinek et al. 2012; Mali, Yang, et al. 2013).
Fig. 8. Structure of CRISPR/Cas9. Cas9 recruitment to the target DNA is

mediated by a chimeric single-guide RNA (sgRNA). It contains a protospacer
recognizing the target sequence followed by protospacer adjacent motif (PAM).
Cas9 protein complexes with sgRNA and binds the target site of genomic DNA,
creating a double-strand break (DSB) at 3 or 4 nucleotides upstream of the PAM
sequence.
31
Upon binding to the target sequence, the Cas9 endonuclease induces specific
double stranded breaks (DSBs), which are repaired by cellular DNA damage
response machinery through two distinct mechanisms: (1) error-prone non-
homologous end joining (NHEJ), the predominant mechanism that results in
stochastic insertions and/or deletions (indels) in the repaired sequence (Jeggo
1998; Rudin, Sugarman, and Haber 1989), or (2) homology-directed repair
(HDR), the less efficient mechanism that results in precise nucleotide edits, since
the break is repaired through the use of an endogenous or exogenous DNA repair
template (K R Thomas, Folger, and Capecchi 1986; Jeggo 1998; Hsu, Lander,
and Zhang 2014; Komor, Badran, and Liu 2017) (Fig. 9). Thus, by inducing
NHEJ or HDR, CRSIPR/Cas9 can be used to delete, replace, or add genetic
sequences.
Fig. 9. CRISPR-Cas9-mediated genome editing. DSB is repaired by Non-homologous end

joining (NHEJ) or Homologous recombination (HR). In NHEJ, random insertions and
deletions are introduced into the genome. In HR, precise mutations are integrated into the target
genomic location by providing a donor sequence that has homology arms with the DSB site.
32
Mechanisms of CRISPR-Cas9 tools

In the CRISPR-Cas9 system, Cas9 is an RNA-guided double-strand DNA
nuclease, and a single guide RNA (sgRNA) directs Cas9 to trigger specific DSBs.
Cas9 nuclease has two catalytic domains: HNH domain that cleaves the target
DNA strand complementary to the 20 nt cRNA and RuvC domain that cleavage
the non-crRNA complementary strand by 3 nt upstream to the PAM site, yielding
DSBs (Jinek et al. 2012) . This enzyme has a highly conserved arginine-rich
region downstream to the region of RuvC domain that is hypothesized to mediate
DNA binding (Sampson et al. 2013). Mutation of aspartate to alanine (D10A) in
RuvC catalytic domain mediates the Cas9 activity to make single stranded breaks
(nickase, Cas9n) rather than DSBs, thus minimize the off-target cleavage (Jinek
et al. 2012). The Cas9n (D10A) can use proper sgRNA to nick both strands,
mediating DSB of the target DNA locus (Ran et al. 2013).
Fig. 10. Cas9 catalytic domains. Cas9 nuclease has two catalytic domains: HNH domain that
cleaves the target DNA strand complementary to the 20 nt cRNA and RuvC domain that
cleavage the non-crRNA complementary strand by 3 nt upstream to the PAM site, yielding
DSBs (Jinek et al. 2012).
33
Mutations H840A and D10A in the HNH and RuvC domains in dCas9,
respectively, abolish cleavage (dCas9) but retain DNA-binding activity. The
dCas9 in conjunction with sgRNA has been developed as gene interference
(CRISPRi) and activation (CRISPRa) tools (Qi et al. 2013; Bikard et al. 2013;
Kiani et al. 2015; Huang et al. 2016; Tong et al. 2015) (Fig. 12, 13 & 14). To
date, CRISPR-Cas tools have been widely applied to edit or regulate genes in
both bacteria such as E. coli (H. Zhang et al. 2017; W. Jiang et al. 2013; Y. Jiang
et al. 2015), Streptococcus pneumoniae (W. Jiang et al. 2013) and Streptomyces
(M. M. Zhang et al. 2017) and fungi such as Aspergillus niger (Sarkari et al. 2017;
Kuivanen, Wang, and Richard 2016).
Fig. 11. Cas9 nuclease (nCas9). The Cas9 nuclease cleaves DNA via its RuvC and HNH
nuclease domains, each of which nicks a DNA strand to generate blunt-end DSBs. Either
catalytic domain can be inactivated to generate nickase mutants that cause single-strand DNA
breaks, thus minimize the off-target cleavage (Jinek et al. 2012).
34
1. CRISPR-Cas9-mediated genome editing
One main application of CRISPR-Cas9 tools is DNA cleavage (Fig.12).

Cas9 is directed by a sgRNA to induce precise DNA cleavage at
endogenous genomic loci. Cas9 can also be mutated into a nicking enzyme
to participate in homology directed repair with minimal mutagenic activity
(Cong et al. 2013). Specially, multiple guide sequences can be aggregated
as a single CRISPR array to enable simultaneous editing of multiple
chromosomal sites (Mans et al. 2015). Presumably, this powerful tool
accelerates strain evolution.
Fig. 12. HDR-mediated targeted editing. HDR, a precise repair mechanism, is capable of generating
gene replacements, gene fusions, gene insertions and gene variants. The HDR process depends on the
simultaneous delivery of a repair template and the generation of double-strand breaks (DSBs).
Different repair outcomes can be generated by manipulating the repair template and homology arms.
Example 1 shows targeted gene replacement in which the entire gene is replaced by a repair template
with homology arms for replacement. Example 2 shows that sequences can be fused to the gene by
supplying a repair template with homology arms suitable for gene fusions. Example 3 shows targeted
gene insertions in which the gene is inserted using a repair template with homology arms to the site
of insertion.
35
2. CRISPR-dCas9-mediated expression control
In addition to DNA cleavage, CRISPR-Cas9 tools exhibit functions of

transcription control, including CRISPRi (Qi et al. 2013) (Fig.14) and
CRISPRa (Fig. 13) (Bikard et al. 2013). The dCas9-sgRNA complex
represses gene expression when dCas9 binds a promoter or an open reading
frame (ORF) (Qi et al. 2013; Bikard et al. 2013). However, this complex
activates gene expression when dCas9 is fused with the omega subunit of
RNA polymerase (Bikard et al. 2013).
2.1. CRISPRa
Catalytically inactive Cas9 (dCas9) is fused with a transcription factor,

which is targeted to upstream of the target gene and delivers the
transcription factor to the promoter, which facilitates the combination of
RNA polymerase (RNAP) and transcription factor for enhancing the
transcriptional efficiency (Qi et al. 2013; Bikard et al. 2013).
Fig. 13. CRISPR activation. Catalytically inactive Cas9 (dCas9) is fused with a transcription
factor, which is targeted to upstream of the target gene and delivers the transcription factor to
the promoter, which facilitates the combination of RNA polymerase (RNAP) and transcription
factor for enhancing the transcriptional efficiency.
36
2. 2. CRISPRi
The dCas9 lacks endonuclease activity, however, when coexpressed with a

guide RNA, the resulting dCas9-gRNA complex can specifically retard RNA
polymerase binding, transcriptional elongation, or transcription factor binding
(Fig. 14). It has been proven that CRISPRi efficiently repressed the expression
of targeted genes in E. coli (Qi et al. 2013), Synechococcus elongates PCC 7942
(Huang et al. 2016), and Actinomycetes [14]. Compared with CRISPR,
CRISPRi is independent of DNA repair mechanism.
Fig. 14. CRISPR interference. There are two ways to silence gene expression. The dCas9
sgRNA complex targets the promoter or enhancer sequence to block the RNAP and/or
transcription factor, the transcription initiation is inhibited. The dCas9-sgRNA complex targets
the gene sequence or its 5' UTR sequence to prevent the transcription elongation.
37
Advantages of CRISPR-Cas9 gene editing tool

The CRISPR/Cas9 system became widely popular, when it was described as a
potent gene-editing technology (Jinek et al. 2012; Mali, Yang, et al. 2013; Cong
et al. 2013). Since then, its research and clinical applications have greatly
surpassed that of other previously established programmable nucleases, including
MNs, ZFNs and TALENs (Boettcher and McManus 2015). To date, more than
10,000 research and review articles have been published on CRISPR/Cas.
Compared to alternative gene-editing technologies, CRISPR/Cas9 provides a
low-cost, highly efficient and simple-to-use gene editing system; it does not
require complex protein design and engineering associated with MNs, ZFNs and
TALENs. Unlike TALENS, CRISPR employs small proteins which are easily
deliverable and unlike ZFNs, it does not require pairs of proteins to target a
specific locus (Adli 2018; Komor et al. 2016). Another significant advantage of
the CRISPR/Cas9 technology is its ability to simultaneously perturb several
genes, with the use of multiple gRNAs targeting different genes (Cong et al. 2013;
H. Wang et al. 2013), opening new possibilities in the study of complex polygenic
disorders.
Table 1. Comparison of CRISPR/Cas9, ZFNs, TALENs and RNAi
CRISPR/Cas9 ZFNs TALENs RNAi

Target site 19–22 bp 18–36 bp 24–40 bp Target site should be
located 50–100 nt from
ATG
Retargeting Easily retargeted Yes, but requires Yes, but requires Yes
possibility without any complex molecular protein engineering
complexity cloning
Nuclease Cas9 FokI FokI Dicer and Argonaute
proteins
Recognition RNA-DNA Protein-DNA Protein-DNA RNA
mechanism
Targeting Protospacer Non-G-rich sequences are T in the start Only targets mRNA
and A at the end
restrictions adjacent motif difficult to target
(PAM) must
be present
Efficiency High High High High
Limitations Off targets Both expensive and time Takes long to Off targets
consuming to construct construct
Cytotoxicity Low Low Variable to high Variable to high
Multiplexing High Low Low High
ease
Cost Low High Moderate Low
38
Delivery systems for CRISPR

Delivery of Cas9 into cells or tissues for therapeutic benefit is an ongoing
challenge. Although initial experiments relied on delivering plasmids or viral
vectors encoding Cas9 and sgRNAs (Howes and Schofield 2015), advances in
non-DNA-dependent options such as preassembled protein–RNA complexes are
opening new delivery avenues. Researchers have explored multiple viral vectors
and delivery methods in order to expand the carrying capacities and the spectrum
of disorders that can be treated.
Viral Delivery Systems

Viral vectors such as adeno-associated virus (AAV), Adenoviral Vectors
(AdV), lentivirus or Bacteriophages are commonly used for delivering genes of
interest in vivo or into cell types resistant to common transfection methods, such
as immune cells. AAV vectors have been commonly used for attractive
candidates for efficient gene delivery in vivo because of their low immunogenic
potential, reduced oncogenic risk from host-genome integration, and well-
characterized serotype specificity. However, the most commonly used Cas9
nuclease encoding gene from Streptococcus pyogenes is >4 kb in length, which
is difficult to transduce using AAV due to its 4.7 kb packaging capacity.
Modification of adenoviral vectors has expanded the carrying capacity to as high
as 35 kb (Parks et al. 1996; D. Palmer and Ng 2003).
However, viral approaches are still highly desirable due to their low
immunogenicity and wide array of characterized tropisms. The size constraints
of viral vectors can be sidestepped by using significantly smaller Cas9 orthologs
derived from metagenomic discovery, several of which have already been
characterized and validated in human cells (Cong et al. 2013; Hou et al. 2013;
Esvelt et al. 2013). Interestingly, short Cas9 variants reported to date recognize
much longer PAM sequences than SpCas9 (5'-NNAGAAW from Streptococcus
thermophilus CRISPR1 or 5'-NNNNGATT from Neisseria meningitidis) (Y.
Zhang et al. 2013; Garneau et al. 2010), whereas some longer orthologs have
more relaxed PAMs (50-NG from Francisella novicida) (Fonfara et al. 2014).
Although the effect of PAM restriction on DNA targeting specificity remains to
be investigated, the more limited overall targeting range of short Cas9 variants
may be partially compensated for by decreasing the number of potential off-target
substrates genome-wide.
39
Table 2. Summary of different delivery vectors.
Non-Viral Delivery
Non-viral delivery methods have also been used to deliver CRISPR/Cas9.
These include naked DNA, DNA encapsulating liposomes, compacted DNA
nanoparticles (e.g., gold, lipid), cell-penetrating peptides and the molecular
Trojan horse (J. Niewoehner et al. 2014). Generally, these methods carry low risk
of immune responses commonly associated with viral delivery (Lino et al. 2018;
Han et al. 2012; Adijanto and Naash 2015). However, due to difficulties in
yielding proper ratio for gene editing and the lack of transduction efficacy, these
methods lack the potential to deliver CRISPR/Cas9 in clinical settings in the near
future (Kabadi et al. 2014).
40
Applications of CRISPR-Cas9 tools

1. Evolution of second-generation CRISPR gene-editing
tools
One of the key progresses in the field of CRISPR technology has been the
development of base-editing technology. Unlike WT Cas9, which results in DSBs
and random indels at the target sites, these so-called second-generation genome
editing tools are able to precisely convert a single base into another without
causing DNA DSBs. The nickase Cas9 is the foundational platform for the base
editor tools that enables direct C to T or A to G conversion at the target site
without DSBs (Gaudelli et al. 2017, 2018; Komor et al. 2016; Nishida et al. 2016).
Komor et al. recently demonstrated that a fusion complex composed of nickase
Cas9 fused to an APOBEC1 deaminase enzyme and Uracyl Glycosylase inhibitor
(UGI) protein effectively converts Cytosine (C) into Thymine (T) at the target
site without causing double strand DNA breaks (Komor et al. 2016). Notably, a
transfer RNA adenosine deaminase has also been evolved and fused to nickase
Cas9 to develop another novel base editor that achieves direct A–G conversion at
the target sites (Gaudelli et al. 2017, 2018). These novel base-editing approaches
significantly expand the scope of genome targeting. Researchers are further
developing these tools for additional purposes.
Fig. 15. Second-generation CRISPR gene editing tools are able to precisely convert a single base
into another without causing DNA DSBs. A fusion complex composed of nickase Cas9 fused to an
APOBEC1 deaminase enzyme and Uracyl Glycosylase inhibitor (UGI) protein effectively converts
Cytosine (C) into Thymine (T) (Komor et al. 2016). A transfer RNA adenosine deaminase has also
been evolved and fused to nickase Cas9 to develop another novel base editor that achieves direct A–G
conversion at the target sites (Gaudelli et al. 2017, 2018).
41
Researchers recently harnessed the efficiency of this CRISPR base editor to

alter genetic code and introduce early STOP codons in genes (Kuscu et al. 2017;
Billon et al. 2017). They show that by editing C into T at CGA (Arg), CAG (Gln),
and CAA (Gln) codons, we can create TGA (opal), TAG (amber), or TAA (ochre)
STOP codons, respectively. The CRISPR-STOP approach is an efficient and less
deleterious alternative to WTCas9-mediated gene knockout (KO) studies (Billon
et al. 2017). In addition to the APOBEC adenosine deaminase enzyme, the
activation-induced adenosine deaminase (AID) enzyme has also been fused to the
dCas9 enzyme (Hess et al. 2016; Y. Ma et al. 2016). Notably, in the absence of
UGI in the complex, the dCas9–AID complex becomes a powerful local
mutagenic agent that acts as a gain of function screening tool (Hess et al. 2016;
Y. Ma et al. 2016; Kuscu and Adli 2016).
42
2. Transcriptional Modulation
dCas9 binding alone to DNA elements may repress transcription by sterically
hindering RNA polymerase machinery (Qi et al. 2013), likely by stalling
transcriptional elongation. This CRISPRi works efficiently in prokaryotic
genomes but is less effective in eukaryotic cells (Gilbert et al. 2013). The
repressive function of CRISPRi can be enhanced by tethering dCas9 to
transcriptional repressor domains such as KRAB or SID effectors, which promote
epigenetic silencing (Gilbert et al. 2013; Konermann et al. 2013). However,
dCas9-mediated transcriptional repression needs to be further improved—in the
current generation of dCas9-based eukaryotic transcription repressors, even the
addition of helper functional domains results in only partial transcriptional
knockdown (Gilbert et al. 2013; Konermann et al. 2013).
Fig. 16. Transcriptional Modulation. A catalytically dead Cas9 (dCas9, containing mutations
in both the RuvC and HNH actives sites) can be used as an RNA-guided DNA binding protein
that can repress both transcription initiation when bound to promoter sequences or transcription
elongation when bound to the template strand within an open reading frame (Bikard et al. 2013;
Qi et al. 2013).
Cas9 can also be converted into a synthetic transcriptional activator by fusing

it to VP16/VP64 or p65 activation domains (Fig. 16). In general, targeting Cas9
activators with a single sgRNA to a particular endogenous gene promoter leads
to only modest transcriptional upregulation (Konermann et al. 2013; Maeder,
Linder, et al. 2013; Perez-Pinera et al. 2013; Mali, Aach, et al. 2013). By tiling a
promoter with multiple sgRNAs, several groups have reported strong synergistic
effects with nonlinear increases in activation (Perez-Pinera et al. 2013; Maeder,
Linder, et al. 2013; Mali, Aach, et al. 2013). Although the requirement for
multiple sgRNAs to achieve efficient transcription activation is potentially
advantageous for increased specificity, screening applications employing
libraries of sgRNAs will require highly efficient and specific transcriptional
control using individual guide RNAs.
43
3. Rapid Generation of Cellular and Animal Models

Genome editing will continue to be used to generate disease models and tissue
donors, and to bridge the gap between therapeutic proof-of-concept validation in
rodents and human clinical trials. The availability of somatic cell nuclear transfer
will enable the study of cardiovascular, immune and metabolic systems in
animals, such as pigs and primates, that mimic human physiology (Klymiuk et al.
2016).
One exciting potential application of CRISPR technologies is to engineer large
animals to study mechanisms of immune rejection and disease transmission
across species barriers. The demonstration last year that endogenous retroviruses
could be eliminated from porcine cells by CRISPR targeting indicates whole
animals could be engineered with a reduced risk of transmitting disease, bringing
xenotransplantation applications one step closer (Yang et al. 2015). For
xenotransplantation to become reality, not only would researchers working with
engineered organs need to address zoonosis concerns, they would also need to
demonstrate effective repression of hyperacute rejection, acute vascular rejection
and chronic cellular xenograft rejection following transplantation across species.
It is conceivable that genome editing could be used to both reduce immune
barriers in the host and improve donor organ function.
CRISPR-mediated genome editing could also expedite the development of
large animal models of human diseases, including in primates, and thereby
accelerate the identification of suitable therapies (Niu et al. 2014), although
mosaicism issues have to be addressed to ensure the genotype of interest is
consistently generated across tissues and cell types. CRISPR-mediated genome
editing is being used to establish robust in vitro and in vivo models of human
disease (Rodolphe Barrangou and May 2015).
4. Epigenetic Control
Complex genome functions are defined by the highly dynamic landscape of
epigenetic states. Epigenetic modifications that tune histones are thus crucial for
transcriptional regulation and play important roles in a variety of biological
functions. These marks, such as DNA methylation or histone acetylation, are
established and maintained in mammalian cells by a variety of enzymes that are
recruited to specific genomic loci either directly or indirectly through scaffolding
proteins.
44
Fig. 17. Epigenetic Control. Cas9 epigenetic effectors (epiCas9s) that can artificially install
or remove specific epigenetic marks at specific loci would serve as a more flexible platform to
probe the causal effects of epigenetic modifications in shaping the regulatory networks of the
genome.
Previously, zinc finger proteins and TAL effectors have been used in a small
number of proof-of-concept studies to achieve locus-specific targeting of
epigenetic modifying enzymes (Beerli, Dreier, and Barbas 2000; Konermann et
al. 2013; Maeder, Angstman, et al. 2013; Mendenhall et al. 2013). Cas9
epigenetic effectors (epiCas9s) that can artificially install or remove specific
epigenetic marks at specific loci would serve as a more flexible platform to probe
the causal effects of epigenetic modifications in shaping the regulatory networks
of the genome (Fig. 17). Of course, the potential for off-target activity or crosstalk
between effector domains and endogenous epigenetic complexes would need to
be carefully characterized. One solution could be to harness prokaryotic
epigenetic enzymes to develop orthogonal epigenetic regulatory systems that
minimize crosstalk with endogenous proteins.
45
5. Large-scale genetic and epigenetic CRISPR screenings

In addition to targeted genetic and epigenetic manipulations, the simple and
efficient gene-targeting capacity of CRISPR has been harnessed to achieve large
scale functional screenings. In such applications, instead of using a single
sgRNA, WT Cas9 or dCas9-effector fusion proteins are guided with hundreds or
thousands of individual sgRNAs in a population of cells. The ultimate aim for
such studies is to identify genes that influence a specific phenotype in an unbiased
fashion (Shalem et al. 2014; T. Wang et al. 2014; Koike-Yusa et al. 2013).
Although the approach requires a number of technical and analytical
considerations (Doench 2017), once established, such an approach becomes a
powerful high throughput assay to functionally screen a large number of genes at
the same time.
In its basic form, a large pool of Cas9/sgRNAs are typically delivered to a
population of cells via a low multiplicity of viral infection (MOI = 0.3 to 0.4).
This ensures that each cell is receiving one or less sgRNA. For robust statistical
readouts, each gene is typically targeted by 6–10 different sgRNAs.
The basic logic behind the CRISPR KO screenings is that if a gene is essential
for a given phenotype, such as cell proliferation, then the cells infected with the
sgRNAs targeting that gene will be relatively depleted from the population over
time. Since each sgRNA is stably integrated into the genome during viral
infection, the guiding sequences of each sgRNA can be used as a unique
‘barcode’. Thus, the relative abundance of each sgRNA in a given population of
cells can be quantified by targeted sequencing.
Previous genome-engineering technologies based on ZFNs and TALENs
required constructing and testing newly designed proteins for each DNA target
sequence to be modified. Therefore, the main advantage of CRISPR technologies
is the ease with which ~80-nt CRISPR sgRNAs can be synthesized to direct Cas9
to different target sequences, which in turn means throughput can be increased.
CRISPR-based screens have enabled identification of essential genes (T. Wang
et al. 2015; Evers et al. 2016) and drug targets (Housden et al. 2015; Parnas et al.
2015). Beyond functional and essential genes, CRISPR-based screening can be
used to study non-coding sequences and characterize enhancer elements and
regulatory sequences (Korkmaz et al. 2016; Rajagopal et al. 2016). This type of
analysis will be crucial to elucidate the roles of the so-called non-coding genome.
46
6. Agricultural applications
Trait improvement through classic breeding in livestock, such as cows,
chickens and pigs, will be accelerated by CRISPR-based genome engineering.
Animal breeders have already identified trait-associated chromosomal markers
known as quantitative trait loci and use marker-assisted breeding to selectively
advance valuable traits. This process will be accelerated using genome editing
technologies, as recently shown in pigs (Whitworth et al. 2015) and dairy cattle
(Carlson 2016), to protect against viruses and remove horns, respectively.
Another application in animals is to engineer production of either medical
products or tissues. For example, knock-in of human albumin cDNA into the pig
Alb locus using CRISPR–Cas9 could enable the production of albumin using
transgenic pigs (J. Peng et al. 2015).
CRISPR-enabled engineering is being used in commercial and model crops to
increase yield, improve drought tolerance and increase growth in limited-nutrient
conditions, and to breed crops with improved nutritional properties (Belhaj et al.
2015; Ricroch and Hénard-Damave 2016). The use of CRISPR technology in
corn (Svitashev et al. 2015) and soybean (Z. Li et al. 2015) illustrates the speed
of adoption of CRISPR technology outside the laboratory. CRISPR-based gene
targeting can also be harnessed to combat plant pathogens, as has been shown for
the tomato yellow leaf curl virus in Nicotiana benthamiana (Ali et al. 2015).
47
7. Applications in food and industrial biotechnology

To date, applications of CRISPR systems in bacteria include genotyping,
vaccinating industrial cultures against viruses, controlling uptake and
dissemination of antibiotic resistance genes by bacteria, and engineering
probiotic cultures (Selle and Barrangou 2015b, 2015a). The commercial success
of native CRISPR–Cas immune systems for the vaccination of Streptococcus
thermophilus starter cultures used in dairy fermentations (yogurt and cheese) has
paved the way for CRISPRs in food (Rodolphe Barrangou et al. 2013; Rodolphe
Barrangou and Horvath 2012). Food-grade applications could be enabled by
screening for natural CRISPR-based vaccination events, which generate oth-
erwise isogenic and iso-functional starter cultures (Rodolphe Barrangou et al.
2013). Recent work has also shown proof of the concept that beneficial bacteria
may be generated that are immunized against the uptake and dissemination of
genes that encode antibiotic resistance (Garneau et al. 2010).
CRISPR technologies will have a broad impact on all industries related to
bacteria, fungi and yeast, as we are on the cusp of the widespread use of genome
editing in these organisms (Oh and van Pijkeren 2014; van Pijkeren and Britton
2014; Rodolphe Barrangou and van Pijkeren 2016). CRISPR–Cas9 is likely to be
used to engineer industrial bacteria, yeast and fungi to manufacture green chemi-
cals, including biofuels (Ryan et al. 2014) and biomaterials. Synthetic biology
approaches that incorporate CRISPR technologies could produce mosaic
genomes that have been streamlined for minimal content by strategic deletions
(Selle and Barrangou 2015a; Selle, Klaenhammer, and Barrangou 2015). A recent
report also showed that CRISPR-mediated vaccination processes can be exploited
as molecular recording events, with the ability to capture synthetic DNA
sequences, useful for data storage, into bacterial, and possibly other, genomes
(Shipman et al. 2016).
48
8. Biological control applications

Cas9 has been used to create gene drives (Esvelt et al. 2014; Gantz et al. 2015;
DiCarlo et al. 2015), in which acquisition of a trait and the Cas9 machinery are
coupled to ensure rapid trait propagation through a population. Specifically, gene
drives can be used in Anopheles gambiae, the mosquito vector for malaria, to
drive a recessive female sterility genotype with transmission to progeny rates
exceeding 90%. Such an approach has the potential to suppress the spread of
malaria in humans (A. Hammond et al. 2015). Likewise, anti-Plasmodium
falciparum CRISPR systems have been implemented in the Asian malaria vector
Anopheles stephensi (Gantz et al. 2015). Notwithstanding the potential of
CRISPR-based gene drives for controlling the spread of disease agents, as with
any nascent technology, successful implementation on a broad scale will require
both scientific advancement (notably regarding biological containment and drive
efficiency), as well as regulatory approval and public acceptance.
49
CRISPR/Cas9 Somatic & Germline editing

Clinical CRISPR/Cas9 applications on somatic cells
Scientists currently estimate that over 10,000 human diseases have monogenic
origin (WHO MEDIA CENTER 2010), the majority of which have no approved
clinical options (“Weblet Importer” n.d.). However, with the breakthrough
development of the CRISPR/Cas9 gene-editing platform, clinical applications of
gene therapy are becoming increasingly conceivable. In fact, months after
CRISPR/Cas9 was first described as a potential gene-editing system (Jinek et al.
2012; Cong et al. 2013; Hsu et al. 2013), three companies were founded, Editas
Medicine, CRISPR Therapeutics and Intellia Therapeutics, aiming at harnessing
the therapeutic potential of this revolutionary technology (Mollanoori and
Teimourian 2018). The number of CRISPR/Cas9 biotechnology companies has
since increased considerably (“The CRISPR Economy: 7 Private Startups
Pursuing The New Frontier In Biotech” n.d.).
Clinical applications of CRISPR/Cas9 gene-editing fall into the two broad
categories of gene therapy: (1) ex vivo approaches, which involve genetic
modification of cells outside of the patient’s body, followed by transplantation of
the modified cells back into the patient, and (2) in vivo approaches, which involve
gene-editing of cells inside the patient’s body and require specialized delivery
systems for the editing machinery (Misra 2013; Zhan et al. 2018). Gene-editing
itself can be sub-categorized based on the type of cells being edited: (1) somatic
cell editing, which includes all non-reproductive cells and does not result in
heritable changes, or, (2) germline editing, performed on embryos, zygotes, or
gametes, and resulting in heritable changes (Sciences, Medicine, and National
Academies of Sciences and Medicine 2017). Human germline editing ranges
from being heavily restricted to being prohibited by law in different countries
(Isasi, Kleiderman, and Knoppers 2016); all current clinical trials involving gene-
editing exclusively target somatic cells, including stem cells.
Current clinical trials

The majority of ongoing clinical trials are employing CRISPR/Cas9 to create
chimeric antigen receptor T cells (CAR-T) for cancer immunotherapies (Zych,
Bajor, and Zagozdzon 2018). Gene-editing of T cells through the CRISPR/Cas9
platform has been extremely successful to date and is broadly recognized as one
50
of the largest leaps in personalized cancer therapeutics (Zych, Bajor, and

Zagozdzon 2018; Cyranoski 2016; Eyquem et al. 2017).
A clinical trial set to start in August 2018 in the United States aims to treat
refractory metastatic gastrointestinal epithelial cancers by administering patient-
derived tumor-infiltrating lymphocytes, which carry CRISPR/Cas9 edited
inactivating mutations in the Cytokine-induced SH2 gene (CISH) (“Home -
ClinicalTrials.Gov” n.d.). Preclinical studies in mice have shown that genetic
deletion of Cish in lymphocytes enhances their expansion and capability to
mediate tumor regression (D. C. Palmer et al. 2015).
Fig. 18. In vivo and ex vivo strategies for CRISPR/Cas9-based gene therapies. (A) In in
vivo approaches, CRISPR/ Cas9 components are directly delivered into the patient using either
viral or non-viral vectors for in situ gene editing. (B) In ex vivo approaches, genes are edited
in patient-derived cells. These can be generated by reprogramming (iPSCs) or direct expansion
of somatic stem/progenitor cells, and are transplanted back into the same patient after the
correction. CRISPR, clustered regularly interspaced short palindromic repeat; iPSCs, induced
pluripotent stem cell.
51
CRISPR therapies in development

Preclinical gene therapies employing CRISPR/Cas9 gene-editing focus on
DMD, Cystic Fibrosis (CF), β-hemoglobinopathies (β-thalassemia, Sickle Cell
Disease) Leber Congenital Amaurosis (LCA) and Primary Hyperoxaluria Type 1
(PH1) (“Home Page | CRISPR” n.d.; “Home - Intellia Therapeutics” n.d.; Dai et
al. 2016; Robinson-Hamm and Gersbach 2016), all of which are heritable
diseases that impact children. CRISPR Therapeutics, Editas Medicine and
Exomics Therapeutics are all developing therapies for DMD (“Weblet Importer”
n.d.; “Home - Exonics Therapeutics” n.d.; “Home Page | CRISPR” n.d.). Using
CRISPR/Cas9, Editas Medicine is planning to restore dystrophin expression by
deleting mutation containing sequences of the gene in vivo, to create smaller but
functional versions of the dystrophin protein (“Weblet Importer” n.d.; Robinson-
Hamm and Gersbach 2016). Exonics Therapeutics, a spin-off company from the
University of Texas Southwestern Medical Center, is also primarily focusing on
DMD treatments using in vivo CRISPR/Cas9 gene-editing technology to repair
exon mutations and restore dystrophin expression (“Home - Exonics
Therapeutics” n.d.), building on results from induced Pluripotent Stem Cells
(iPSCs) and mouse studies (Long et al. 2016). Editas Medicine is additionally
focusing on developing locally injectable CRISPR/Cas9-based therapies into the
eye for LCA, the most prevalent cause of inherited childhood blindness, which
currently has no treatment (“Weblet Importer” n.d.). CRISPR/Cas9 will be
originally used to target the most common mutations responsible for the disease,
found in the CEP290 gene, with the goal of restoring CEP290 protein expression
and improving photoreceptor cell function before the disease progresses to vision
loss (“Weblet Importer” n.d.).
Both β-thalassemia and Sickle Cell Disease (SCD) arise through mutations in
the β-globin gene (Dever et al. 2016). CRISPR Therapeutics is developing
CTX001 for the treatment of both conditions, building on reports of cases
demonstrating that persistence of the infant version of hemoglobin (HBF) in
adults confers protective effects for β-thalassemia or SCD patients (Musallam et
al. 2012, 2013). The premise behind CTX001 is to use gene-editing on patients’
bone marrow cells ex vivo to induce production of HBF (“Home Page | CRISPR”
n.d.). CRISPR Therapeutics recently initiated the first CTX001 clinical trial in
Europe, employing CRISPR/Cas9 edited CD34+ hHSPCs for the treatment of β-
thalassemia (“Clinical Trials Register” n.d.). A request for authorization for a
CTX001 clinical trial in the United States has been filed to the FDA, but approval
52
is still pending (“FDA Places CTX001 for SCD on Clinical Hold | MDedge
Hematology and Oncology” n.d.). An alternative approach for the treatment of β
hemoglobinopathies includes utilizing CRISPR/Cas9 gene-editing to correct the
causative mutations on the β-globin gene in iPSCs or Hematopoietic Stem Cells
(HSCs) ex vivo (Porteus 2017). Finally, Intellia Therapeutics is developing an in
vivo CRISPR/Cas9 gene-editing treatment of PH1, a life-threatening condition
characterized by recurrent kidney and bladder stones due to increased oxalate
production (Lorenzo-Sellares et al. 2014); a detailed targeting strategy has not
been made public (“Home - Intellia Therapeutics” n.d.).
CRISPR/Cas9 germline editing and heritable changes

Somatic cell gene-editing, discussed in the previous sections, exclusively
influences the cells whose genome is being modified, and as such, carries
minimal risk of affecting future generations (Sciences, Medicine, and National
Academies of Sciences and Medicine 2017). Gene-editing of the germline
(gametes, zygotes and embryos) however, can result in propagation of the genetic
modification in every cell of the edited individual (Sciences, Medicine, and
National Academies of Sciences and Medicine 2017). Since reproductive cells
are also affected, the introduced genetic changes would be transmitted to the
offspring of the edited individual (Sciences, Medicine, and National Academies
of Sciences and Medicine 2017). This application of gene therapy has the
potential to eradicate heritable genetic diseases, however, social, ethical and
religious concerns have kept research on human germline gene-editing strictly
regulated, while clinical applications are currently prohibited in more than 50
countries around the world (“Welcome to CGS | Center for Genetics and Society”
n.d.). Considering the pace at which genome editing technologies are evolving,
the scientific world has requested that a global moratorium be placed on the
clinical applications of such technologies, until a comprehensive regulatory
framework is built to provide strict oversight and risk/benefit assessments
(Sciences, Medicine, and National Academies of Sciences and Medicine 2017;
Baltimore et al. 2015).
53
CRISPR/Cas9 on non-viable human zygotes

To date, six studies have been reported utilizing CRISPR/Cas9 technology in
germline editing of human embryos (Kang et al. 2016; Liang et al. 2015; H. Ma
et al. 2017; Tang et al. 2017; G. Li et al. 2017; Zhou et al. 2017), five from
research institutes in China and one from the United States. The first two studies
considered ethical concerns and utilized non-viable triploid (tripronuclear, 3PN)
zygotes, which carry one oocyte nucleus and two sperm nuclei and are discarded
during In Vitro Fertilization (IVF). These 3PN zygotes can generate blastocysts
but fail to develop normally in vivo (Liang et al. 2015; Kang et al. 2016). Liang
and colleagues were the first to apply CRISPR/Cas9 gene-editing in human
embryos, to correct β-thalassemia causing mutations on the β-globin gene (Liang
et al. 2015). Their results demonstrated moderate cleavage efficiency by Cas9,
repair predominantly through NHEJ, off-target effects and mosaicism (Liang et
al. 2015). Additionally, of the few HDR-edited embryos, most were repaired with
the δ-globin gene as a template (high sequence similarity with the β-globin gene)
instead of the exogenous DNA oligonucleotide, resulting in mutations in the
edited β-globin gene (Liang et al. 2015).
Kang and colleagues used the same technology to introduce a naturally
occurring deletion of the C-C chemokine receptor type 5, a major co-receptor of
HIV-1, in 3PN embryos (Kang et al. 2016). This study demonstrated high
efficiency of Cas9 cleavage and no offtarget effects, based on a limited number
of predicted off-target sites (Kang et al. 2016). Preferential repair through NHEJ
was consistently reported, however in this case it proved advantageous towards
introducing the desired CCR5Δ32 variant in the embryos (Kang et al. 2016).
Mosaicism was also present (Kang et al. 2016).
Two further studies utilizing human 3PN zygotes employed CRISPR base
editors to introduce known mutations in wild type zygotes to test the technology
(G. Li et al. 2017; Zhou et al. 2017). High editing efficiency at the target sites
was consistently observed (G. Li et al. 2017; Zhou et al. 2017), however Zhou
and colleagues reported moderate incidence of off-target effects, including indels
and editing of bases around the target sites (Zhou et al. 2017).
54
CRISPR/Cas9 on viable human zygotes

To date, two studies have demonstrated CRISPR editing of normal (dual
pronuclear, 2PN) human zygotes (H. Ma et al. 2017; Tang et al. 2017). Tang and
colleagues applied CRISPR/Cas9 gene-editing to correct point mutations in
Glucose-6 Phosphate Dehydrogenase (G6PD) or the β-globin gene (Tang et al.
2017). High Cas9 cleavage efficiency and improved HDR specific repair was
reported for both G6PD and β-globin gene-editing, however the authors used a
very small number of zygotes per experiment, and since roughly half of these
were wild type for the mutations, statistical conclusions cannot be drawn (Tang
et al. 2017). Mosaicism was consistently present, while off-target effects were not
detected in the one correctly edited embryo for G6PD that was examined (Tang
et al. 2017). An additional interesting finding from the Tang study was that the
use of Cas9 protein instead of mRNA increased the incidence of HDR repair in
3PN zygotes (these were used during pilot experiments optimizing the
technology), although it remained considerably lower than NHEJ (Tang et al.
2017).
Most recently, Ma and colleagues used CRISPR/Cas9 to correct a myosin
binding protein C (MYBPC) mutation known to cause hypertrophic
cardiomyopathy (H. Ma et al. 2017). By injecting 2PN zygotes they showed high
Cas9 cleavage efficiency and increased HDR-specific repair compared to
previous studies, however embryonic mosaicism remained high (H. Ma et al.
2017). To address this issue, CRISPR/Cas9 was mixed with sperm and the
mixture was co-injected into oocytes during Intracytoplasmic Sperm Injection
(ICSI) (H. Ma et al. 2017). This method dramatically reduced embryo mosaicism,
while it further increased cleavage efficiency and HDR-specific repair (H. Ma et
al. 2017). Comprehensive analysis did not yield any off-target effects in zygote
or ICSI edited embryos (H. Ma et al. 2017). Additionally, the authors concluded
that in both zygote and ICSI edited embryos, HDR was predominantly performed
with the homologous maternal wild type allele as a template, instead of the co
injected DNA oligonucleotide (H. Ma et al. 2017).
55
Challenges of CRISPR-Cas9 applications

So far, the applications of CRISPR-Cas9 tools still face limitations. These
setbacks include the lack of methods for generation and delivery of a sgRNA
array, low efficiency of DNA repair in nonmodel microorganisms, limited
strategies to couple with other genetic tools, and off-target effects.
1. Difficulty in generation and delivery of sgRNA array

Since metabolite production relies heavily on coordinated expression of
multiple genes, a gRNA array is required to simultaneously direct Cas9/dCas9 to
defined sites for editing, activating or repressing genes. So far, the multiplex
editing efficiency of the CRISPR-Cas9 system remains to be constrained by the
gRNA expressing device (Nowak et al. 2016). To address this, Xie et al.
developed a general strategy and platform for precise processing and efficient
production of numerous gRNAs in vivo from a synthetic polycistronic gene via
the endogenous tRNA-processing system (Xie, Minkenberg, and Yang 2015).
This strategy is shown to significantly enhance CRISPR/Cas9 multiplex editing
efficiency and has broad applications for small RNA expression and genome
engineering. Apart from the generation of the sgRNA library, the delivery of
CRISPR-Cas9 to intended genetic loci is also vital for efficient genome
engineering. To address this concern, Xu et al. harnessed the piggyback
transposon as a method to deliver a gRNA library for in vivo screening (C. Xu et
al. 2017). Given the availability of transposon systems (Sharpe et al. 2007), we
believe that they could be feasible vehicles for the navigation of gRNA array in
cells.
2. Low efficiency of DNA repair system

In eukaryotes, double-strand DNA breaks (DSBs) are repaired by NHEJ
mechanism in an error-prone manner, leading to insertion/ deletion mutations that
frequently manifest frame shifts and gene disruption. In contrast to eukaryotic
genomes that all contain NHEJ like systems, only a percentage of prokaryotic
genomes harbor NHEJ machinery (Ku/LigD) (Bowater and Doherty 2006). In
fact, bacteria mainly rely on HR to repair DNA breaks (Datta et al. 2008). Several
studies have shown that coupling recombineering with Cas9 counterselection
facilitates genome editing. These topics involve four aspects: (1) single-strand
DNA recombineering (SSDR); (2) double-strand DNA recombineering (DSDR);
56
(3) non-recombineering-based homologous recombination (NrHR); and (4)

NHEJ.
(1) Single-Strand DNA Recombineering (SSDR)

The first SSDR-CRISPR-Cas9-based genome editing tool was reported in
2013 (W. Jiang et al. 2013). E. coli was transformed with the vector pCas9 for
heterologous expression of SpyCas9 and the tracrRNA molecule in order to edit
its genome. E. coli was simultaneously transformed with a linear ssDNA
oligonucleotide for SSDR and with the pCRISPR vector. The pCRISPR vector
harbored a CRISPR array with one spacer that targeted the gene of interest, which
lead to a CRISPR-Cas9- based counterselection against the wild-type genes. This
tandem arrangement of an SSDR system followed by CRISPR-Cas9-based
counterselection constitutes an efficient genome editing tool (W. Jiang et al.
2013). A similar tool was developed for Lactobacillus reuteri (Oh and
van Pijkeren 2014). This tool was based on a SpyCas9 and tracrRNA expressing
vector and the strain L. reuteri 6475, which expresses the phage-derived
ssDNAbinding protein RecT required for SSDR. The SpyCas9 and tracrRNA
expressing strain L. reuteri 6475 were simultaneously transformed with a
CRISPR array expressing vector and ssDNA recombineering fragments. When
no homologous template was supplied, transformation with targeting Cas9
constructs failed to generate transformants, indicating the lethality of using Cas9
(Oh and van Pijkeren 2014). Moreover, scientists realized that coupling CRISPR-
Cas9 system with lambda Red recombineering allows not only highly efficient
recombination of short single-strand oligonucleotides but also replacement of
long DNA fragment (Pyne et al. 2015).
(2) Double-Strand DNA Recombineering (DSDR)

A DSDR-CRISPR-Cas9-based counter selection editing system was also
developed for E. coli (Y. Jiang et al. 2015). This system relies on a λ-Red and
SpyCas9 expressing vector, and a gRNA expressing plasmid. When dsDNA
fragments were used as editing templates, the single gene deletion efficiency was
comparable to the efficiency previously reported (W. Jiang et al. 2013); however,
the gene insertion efficiency was low and proportional to the length of the
homologous regions when using the HR template fragments. In the same study,
a variant of this tool was developed, based on a SpyCas9 and λ-Red expressing
plasmid, and a sgRNA expressing plasmid containing a HR template (Y. Jiang et
al. 2015). The editing tool was used for the simultaneous insertion of two genes
57
or the deletion of three genes in E. coli and the deletion of two genes from the
chromosome of Tatumella citrea (Y. Jiang et al. 2015). Even though this tool was
not based on a classic DSDR system, as the provided editing templates were
plasmid-borne and not linear dsDNA fragments, it is interesting that the
efficiency of this tool dropped in the absence of a functional λ-Red mechanism.
(3) Non-recombineering-based Homologous Recombination

(NrHR)
Since NHEJ-dependent repair of DNA double-strand breaks (DSBs) gives rise
to unwanted insertions or deletions, DNA-nicking enzyme (nickase) was
employed to generate single-strand breaks (SSBs) instead of DSBs, which can be
repaired by error-free HR rather than mutagenic NHEJ. For example, since Cas9-
induced DSBs are lethal to Clostridium cellulolyticum due to weak expression of
NHEJ components, Cas9 nickase, an enzyme causing singlestranded breaks in
duplex DNA, was applied to achieve singlenick-triggered HR that allows one-
step editing at intended genomic loci (T. Xu et al. 2015) . In addition, an NrHR-
CRISPR-Cas9 based counterselection editing tool was developed to target
insertion sequences native to E. coli genome, leading to large chromosomal
deletions (Standage-Beier, Zhang, and Wang 2015). This approach addresses the
concern of CRISPRCas9-induced lethal DSBs, and may work efficiently in the
genome editing of prokaryotes that lack well-developed recombineering systems.
(4) Non-Homologous End Joining (NHEJ)

Tong et al. developed a CRISPR-Cas9-NHEJ tool in Actinomycetales. When
no templates for homology-directed repair (HDR) were present, the site-specific
DNA DSBs introduced by Cas9 were repaired through the error-prone NHEJ
pathway. The resulting deletions were of variable sizes surrounding around the
targeted sequence (Table 1). However, if templates for HDR were supplied at the
same time, precise deletions of the targeted gene were achieved (Tong et al.
2015). Clearly, in all cases, highly efficient DNA repair system is critical for
CRISPR-assisted recombineering.
58
3. Arrangements of CRISPR-Cas system in chromosome

or plasmid
Vector-dependent overexpression of key enzymes is a common strategy for
directing carbon flux to desired pathways. However, this strategy shows its
drawbacks by hindering cell growth and overconsuming cellular resources.
Ideally, Cas9 or dCas9 is inserted into chromosome and subjected to induction
expression. By contrast, sgRNAs should be user-defined since its expression
device is vector borne or located in the chromosome depending on certain
requirements. For instance, a CRISPR-Cas9 toolkit was developed for the
engineering of Bacillus subtilis by chromosomal expression of Cas9 and
chromosomal transcription of gRNAs using a gRNA transcription cassette and
counterselectable gRNA delivery vectors. This design avoids the need for
multicopy plasmids which can be unstable and impede cell viability (Westbrook,
Moo-Young, and Chou 2016).
4. Coupling CRISPR-Cas9 with other genetic tools

CRISPR tools are versatile but will not work well for all genetic engineering
purposes. For instance, the generation of Cas9 relies on translation machinery and
therefore consumes more resources when compared to RNAi where no translation
process is involved. Clearly, RNAi remains a cost-effective strategy for
transcription control. Additionally, while the Cas9-based gene editing system
relies on repair of DNA breaks in prokaryotic cells, both antisense RNA and
CRISPRi are independent of any DNA repair system. More interestingly, Lee et
al. demonstrated that the gene target repressed by the CRISPR system can be
undone by expressing an antisense RNA that sequesters a small guide RNA (Lee
et al. 2016). Hence, the combination of CRISPR tools with RNAi or antisense
RNA approach could be a clever solution to multidimensional control of gene
expression.
59
5. Off-target effects
CRISPR/Cas9 can trigger off-target mutations and chromosomal
rearrangements, which may alter microbial phenotypes. For rational-design strain
engineering, off target effects are not desirable. Given that off-target effects are
ascribed mainly to sgRNA, unique target sequences should be different from any
other sites in the genome by at least three nucleotides in 20-nucleotide sequences.
Many web-based tools and online software were developed to predict off-targets
and to improve gRNA design. In principle, sgRNA sequence should match
transcription initiation site especially that in non-template strand (Qi et al. 2013).
In addition, sgRNA sequence can be used as a query to search against genome to
avoid targeting homologous sequence. So far, off-target effects remain the major
limitation for CRISPR applications. Thus, in-depth exploration of the underlying
mechanisms is required.
60
CRISPR and The Ethical dilemma

Like most paradigm-shifting technologies, CRISPR is a double-edged sword.
Its gene editing technology has the potential to deliver significant improvements
to human health and wellbeing, but if not deployed responsibly, CRISPR could
also do great harm. It could increase inequality, perpetuate prejudices, jeopardize
health, and even threaten biosecurity. Decisions about how CRISPR should be
used must account for multiple (and sometimes competing) societal values.
Navigating the transformative complexity of CRISPR and steering its course to
maximize benefits and minimize associated risks will be no easy feat. Wise use
of CRISPR is an issue that should be examined through a broad lens, informed
by a diversity of knowledge and perspectives (Memi, Ntokou, and Papangeli
2018).
Contemplating the overall impact of the CRISPR/Cas9 technology, Mulvihill
and colleagues argued that: “CRISPR’s greatest contribution will surely be the
sheer pace, depth, and breath of applications and findings it permits” (Mulvihill
et al. 2017). Considering CRISPR through an ethical lens, however, the
development of this technology has not fundamentally changed the ethical issues
surrounding gene therapy and genetic engineering, except perhaps, that it has
brought these issues forward for immediate consideration.
The ethics of human applications of gene-editing are broadly discussed within
two categories, namely genetic modifications aiming to correct faulty genes (gene
therapy), and modifications that focus on improvement of physiologically normal
genes (genetic enhancement) (Sýkora 2018). Ethical controversy additionally
arises on the topic of the type of cells that are being gene-edited, somatic or
germline (Sýkora 2018). Gene therapy applied to somatic cells is generally
considered ethically sound, since the impacts would be contained to a single
patient, and not passed down to future generations. However, genetic
enhancement of somatic cells and gene therapy or genetic enhancement of
germline cells encompass varying degrees of controversy; enhancement of the
germline is generally considered to be immoral. In fact, article 24 of the Universal
Declaration on the Human Genome and Human Rights that was adopted by the
General Conference of the United Nations Educational, Scientific and Cultural
Organization (UNESCO) in 1997 states that germline modifications “could be
contrary to human dignity” (UNESCO 1997).
61
From current literature on the topic, one can divide the ethical controversy of
germline gene-editing into three central concepts/dilemmas (Sciences, Medicine,
and National Academies of Sciences and Medicine 2017; De Miguel Beriain and
Marcos del Cano 2018), which will be discussed here individually. (1) “Necessity
of germline gene-editing”.
A second pillar of germline gene-editing ethics is (2) “unforeseeable/
unidentified risks in germline gene-editing”. The primary risks involved with the
CRISPR/Cas9 technology include targeting efficiency, off-target effects and
immunogenicity, as discussed previously. According to Baltimore and
colleagues: “As with any therapeutic strategy, higher risks can be tolerated when
the reward of success is high, but such risks also demand higher confidence in
their likely efficacy” (Baltimore et al. 2015).
Finally, a hypothesis that has been around since the conception of gene therapy
argues the existence of a (3) “slippery slope” in germline gene editing. This refers
to the potential use of gene-editing technologies towards eugenics (De Miguel
Beriain and Marcos del Cano 2018), and is founded on the unclear demarcation
between therapy and enhancement (Sciences, Medicine, and National Academies
of Sciences and Medicine 2017). This theory is generally considered farfetched
by scientists; desirable traits including intelligence, confidence, sociability or
even physical appearance are complex, polygenic and are often influenced by the
environment (Hershlag and Bristow 2018). However, the simplicity and efficacy
of the CRISPR/Cas9 technology has already yielded results of genetic
enhancement in research, farm and pet animals (Burkard et al. 2017; Zheng et al.
2017; Cyranoski 2015; Zou et al. 2015).
62
Legislation and logistical considerations

Legislation on human germline editing varies among different countries,
ranging from complete ban on germline modification to intermediate or
permissive policies that are meant to support scientific progress, with appropriate
oversight (Isasi, Kleiderman, and Knoppers 2016). The United States allows for
some research applications of human germline editing, although federal funding
is not available for human embryo research. Several countries additionally
prohibit in vitro culture of human embryos beyond the 14th day of development,
which is approximately the stage of primitive streak emergence (Hyun,
Wilkerson, and Johnston 2016). The central arguments behind the 14-day rule
included the absence of nervous system specification, the possibility of embryo
twinning, the lack of implantation and the observed substantial embryo loss until
this timepoint (Pera 2017). However, when these reports were published, culture
of human embryos beyond a few days post fertilization was practically impossible
(Cavaliere 2017). Recently, two different laboratories reported culturing human
embryos until the 13th day post fertilization (Deglincerti et al. 2016; Shahbazi et
al. 2016), rekindling the debate for revisiting the 14-day rule. These policies,
although not uniformly consistent throughout the world, impose clear barriers on
research involving human germline gene-editing, including CRISPR/Cas9
applications.
63
References
Adijanto, Jeffrey, and Muna I Naash. 2015. “Nanoparticle-Based Technologies for Retinal
Gene Therapy.” European Journal of Pharmaceutics and Biopharmaceutics 95: 353–67.
https://doi.org/https://doi.org/10.1016/j.ejpb.2014.12.028.
Adli, Mazhar. 2018. “The CRISPR Tool Kit for Genome Editing and Beyond.” Nature
Communications 9 (1): 1911. https://doi.org/10.1038/s41467-018-04252-2.
Agrawal, Neema, P V N Dasaradhi, Asif Mohmmed, Pawan Malhotra, Raj K Bhatnagar, and
Sunil K Mukherjee. 2003. “RNA Interference: Biology, Mechanism, and Applications.”
Microbiology and Molecular Biology Reviews 67 (4): 657 LP-685.
https://doi.org/10.1128/MMBR.67.4.657-685.2003.
Ali, Zahir, Aala Abul-faraj, Lixin Li, Neha Ghosh, Marek Piatek, Ali Mahjoub, Mustapha Aouida,
et al. 2015. “Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9
System.” Molecular Plant 8 (8): 1288–91. https://doi.org/10.1016/j.molp.2015.02.011.
Amitai, Gil, and Rotem Sorek. 2016. “CRISPR–Cas Adaptation: Insights into the Mechanism of
Action.” Nature Reviews Microbiology 14 (2): 67–76.
https://doi.org/10.1038/nrmicro.2015.14.
Anders, Carolin, Ole Niewoehner, Alessia Duerst, and Martin Jinek. 2014. “Structural Basis of
PAM-Dependent Target DNA Recognition by the Cas9 Endonuclease.” Nature 513 (7519):
569–73. https://doi.org/10.1038/nature13579.
Arslan, Zihni, Veronica Hermanns, Reinhild Wurm, Rolf Wagner, and Ümit Pul. 2014.
“Detection and Characterization of Spacer Integration Intermediates in Type I-E CRISPR–
Cas System.” Nucleic Acids Research 42 (12): 7884–93.
http://dx.doi.org/10.1093/nar/gku510.
Babu, Mohan, Natalia Beloglazova, Robert Flick, Chris Graham, Tatiana Skarina, Boguslaw
Nocek, Alla Gagarinova, et al. 2011. “A Dual Function of the CRISPR–Cas System in
Bacterial Antivirus Immunity and DNA Repair.” Molecular Microbiology 79 (2): 484–502.
https://doi.org/10.1111/j.1365-2958.2010.07465.x.
Baltimore, David, Paul Berg, Michael Botchan, Dana Carroll, R Alta Charo, George Church,
Jacob E Corn, et al. 2015. “A Prudent Path Forward for Genomic Engineering and
Germline Gene Modification.” Science 348 (6230): 36 LP-38.
https://doi.org/10.1126/science.aab1028.
Barrangou, R., C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D. A. Romero, and
P. Horvath. 2007. “CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes.”
Science 315 (5819): 1709–12. https://doi.org/10.1126/science.1138140.
Barrangou, Rodolphe, Anne-Claire Coûté-Monvoisin, Buffy Stahl, Isabelle Chavichvily, Florian
Damange, Dennis A. Romero, Patrick Boyaval, Christophe Fremaux, and Philippe
Horvath. 2013. “Genomic Impact of CRISPR Immunization against Bacteriophages.”
Biochemical Society Transactions 41 (6): 1383 LP-1391.
https://doi.org/10.1042/BST20130160.
64
Barrangou, Rodolphe, and Philippe Horvath. 2012. “CRISPR: New Horizons in Phage
Resistance and Strain Identification.” Annual Review of Food Science and Technology 3
(1): 143–62. https://doi.org/10.1146/annurev-food-022811-101134.
Barrangou, Rodolphe, and Andrew P May. 2015. “Unraveling the Potential of CRISPR-Cas9 for
Gene Therapy.” Expert Opinion on Biological Therapy 15 (3): 311–14.
https://doi.org/10.1517/14712598.2015.994501.
Barrangou, Rodolphe, and Jan-Peter van Pijkeren. 2016. “Exploiting CRISPR–Cas Immune
Systems for Genome Editing in Bacteria.” Current Opinion in Biotechnology 37: 61–68.
https://doi.org/https://doi.org/10.1016/j.copbio.2015.10.003.
Beerli, Roger R, Birgit Dreier, and Carlos F Barbas. 2000. “Positive and Negative Regulation of
Endogenous Genes by Designed Transcription Factors.” Proceedings of the National
Academy of Sciences 97 (4): 1495 LP-1500. https://doi.org/10.1073/pnas.040552697.
Belhaj, Khaoula, Angela Chaparro-Garcia, Sophien Kamoun, Nicola J Patron, and Vladimir
Nekrasov. 2015. “Editing Plant Genomes with CRISPR/Cas9.” Current Opinion in
Biotechnology 32: 76–84.
https://doi.org/https://doi.org/10.1016/j.copbio.2014.11.007.
Beloglazova, Natalia, Greg Brown, Matthew D Zimmerman, Michael Proudfoot, Kira S
Makarova, Marina Kudritska, Samvel Kochinyan, et al. 2008. “A Novel Family of
Sequence-Specific Endoribonucleases Associated with the Clustered Regularly
Interspaced Short Palindromic Repeats.” Journal of Biological Chemistry 283 (29):
20361–71. https://doi.org/10.1074/jbc.M803225200.
Beloglazova, Natalia, Konstantin Kuznedelov, Robert Flick, Kirill A Datsenko, Greg Brown, Ana
Popovic, Sofia Lemak, Ekaterina Semenova, Konstantin Severinov, and Alexander F
Yakunin. 2015. “CRISPR RNA Binding and DNA Target Recognition by Purified Cascade
Complexes from Escherichia Coli.” Nucleic Acids Research 43 (1): 530–43.
Benda, Christian, Judith Ebert, Richard A. Scheltema, Herbert B. Schiller, Marc Baumgärtner,
Fabien Bonneau, Matthias Mann, and Elena Conti. 2014. “Structural Model of a CRISPR
RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4.” Molecular
Cell 56 (1): 43–54. https://doi.org/https://doi.org/10.1016/j.molcel.2014.09.002.
Bernstein, Emily, Amy A Caudy, Scott M Hammond, and Gregory J Hannon. 2001. “Role for a
Bidentate Ribonuclease in the Initiation Step of RNA Interference.” Nature 409 (January):
363. https://doi.org/10.1038/35053110.
Bhakta, Mital S, Isabelle M Henry, David G Ousterout, Kumitaa Theva Das, Sarah H Lockwood,
Joshua F Meckler, Mark C Wallen, et al. 2013. “Highly Active Zinc-Finger Nucleases by
Extended Modular Assembly.” Genome Research 23 (3): 530–38.
https://doi.org/10.1101/gr.143693.112.
Bikard, David, Wenyan Jiang, Poulami Samai, Ann Hochschild, Feng Zhang, and Luciano A
Marraffini. 2013. “Programmable Repression and Activation of Bacterial Gene
Expression Using an Engineered CRISPR-Cas System.” Nucleic Acids Research 41 (15):
7429–37. http://dx.doi.org/10.1093/nar/gkt520.
65
Billon, Pierre, Eric E Bryant, Sarah A Joseph, Tarun S Nambiar, Samuel B Hayward, Rodney
Rothstein, and Alberto Ciccia. 2017. “CRISPR-Mediated Base Editing Enables Efficient
Disruption of Eukaryotic Genes through Induction of STOP Codons.” Molecular Cell 67
(6): 1068–1079.e4. https://doi.org/10.1016/j.molcel.2017.08.008.
Boch, Jens, Heidi Scholze, Sebastian Schornack, Angelika Landgraf, Simone Hahn, Sabine Kay,
Thomas Lahaye, Anja Nickstadt, and Ulla Bonas. 2009. “Breaking the Code of DNA
Binding Specificity of TAL-Type III Effectors.” Science 326 (5959): 1509 LP-1512.
https://doi.org/10.1126/science.1178811.
Boettcher, Michael, and Michael T. McManus. 2015. “Choosing the Right Tool for the Job:
RNAi, TALEN, or CRISPR.” Molecular Cell 58 (4): 575–85.
https://doi.org/10.1016/j.molcel.2015.04.028.
Bogdanove, Adam J, and Daniel F Voytas. 2011. “TAL Effectors: Customizable Proteins for DNA
Targeting.” Science 333 (6051): 1843 LP-1846.
Bolotin, Alexander, Benoit Quinquis, Alexei Sorokin, and S. Dusko Ehrlich. 2005. “Clustered
Regularly Interspaced Short Palindrome Repeats (CRISPRs) Have Spacers of
Extrachromosomal Origin.” Microbiology 151 (8): 2551–61.
https://doi.org/10.1099/mic.0.28048-0.
Bondy-Denomy, Joe, April Pawluk, Karen L. Maxwell, and Alan R. Davidson. 2012.
“Bacteriophage Genes That Inactivate the CRISPR/Cas Bacterial Immune System.” Nature
493 (7432): 429–32. https://doi.org/10.1038/nature11723.
Bondy-Denomy, Joseph, Bianca Garcia, Scott Strum, Mingjian Du, MaryClare F. Rollins, Yurima
Hidalgo-Reyes, Blake Wiedenheft, Karen L. Maxwell, and Alan R. Davidson. 2015.
“Multiple Mechanisms for CRISPR–Cas Inhibition by Anti-CRISPR Proteins.” Nature 526
(7571): 136–39. https://doi.org/10.1038/nature15254.
Bowater, Richard, and Aidan J Doherty. 2006. “Making Ends Meet: Repairing Breaks in
Bacterial DNA by Non-Homologous End-Joining.” PLOS Genetics 2 (2): e8.
https://doi.org/10.1371/journal.pgen.0020008.
Brouns, Stan J J, Matthijs M Jore, Magnus Lundgren, Edze R Westra, Rik J H Slijkhuis,
Ambrosius P L Snijders, Mark J Dickman, Kira S Makarova, Eugene V Koonin, and John
van der Oost. 2008. “Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes.” Science
321 (5891): 960 LP-964. https://doi.org/10.1126/science.1159689.
Burkard, Christine, Simon G Lillico, Elizabeth Reid, Ben Jackson, Alan J Mileham, Tahar Ait-Ali,
C Bruce A Whitelaw, and Alan L Archibald. 2017. “Precision Engineering for PRRSV
Resistance in Pigs: Macrophages from Genome Edited Pigs Lacking CD163 SRCR5 Domain
Are Fully Resistant to Both PRRSV Genotypes While Maintaining Biological Function.”
PLOS Pathogens 13 (2): 1–28. https://doi.org/10.1371/journal.ppat.1006206.
Cady, Kyle C, Joe Bondy-Denomy, Gary E Heussler, Alan R Davidson, and G. A. O’Toole. 2012.
“The CRISPR/Cas Adaptive Immune System of Pseudomonas Aeruginosa Mediates
Resistance to Naturally Occurring and Engineered Phages.” Journal of Bacteriology 194
(21): 5728–38. https://doi.org/10.1128/JB.01184-12.
66
Carlson, Robert. 2016. “Estimating the Biotech Sector’s Contribution to the US Economy.”
Nature Biotechnology 34 (March): 247–55. https://doi.org/doi.org/10.1038/nbt.3491.
Carmell, M A, Z Xuan, M Q Zhang, and G J Hannon. 2002. “The Argonaute Family: Tentacles
That Reach into RNAi, Developmental Control, Stem Cell Maintenance, and
Tumorigenesis.” Genes Dev 16. https://doi.org/10.1101/gad.1026102.
Carte, Jason, Ross T Christopher, Justin T Smith, Sara Olson, Rodolphe Barrangou, Sylvain
Moineau, Claiborne V C Glover III, Brenton R Graveley, Rebecca M Terns, and Michael P
Terns. 2014. “The Three Major Types of CRISPR-Cas Systems Function Independently in
CRISPR RNA Biogenesis in Streptococcus Thermophilus.” Molecular Microbiology 93 (1):
98–112. https://doi.org/10.1111/mmi.12644.
Carthew, Richard W, and Erik J Sontheimer. 2009. “Origins and Mechanisms of MiRNAs and
SiRNAs.” Cell 136 (4): 642–55. https://doi.org/10.1016/j.cell.2009.01.035.
Catalanotto, Caterina, Gianluca Azzalin, Giuseppe Macino, and Carlo Cogoni. 2000. “Gene
Silencing in Worms and Fungi.” Nature 404 (March): 245.
https://doi.org/10.1038/35005169.
Cavaliere, Giulia. 2017. “A 14-Day Limit for Bioethics: The Debate over Human Embryo
Research.” BMC Medical Ethics 18 (1): 38. https://doi.org/10.1186/s12910-017-0198-5.
Cermak, Tomas, Erin L Doyle, Michelle Christian, Li Wang, Yong Zhang, Clarice Schmidt, Joshua
A Baller, Nikunj V Somia, Adam J Bogdanove, and Daniel F Voytas. 2011. “Efficient Design
and Assembly of Custom TALEN and Other TAL Effector-Based Constructs for DNA
Targeting.” Nucleic Acids Research 39 (12): e82–e82.
http://dx.doi.org/10.1093/nar/gkr218.
Cerutti, Heriberto, and J Armando Casas-Mollano. 2006. “On the Origin and Functions of RNA-
Mediated Silencing: From Protists to Man.” Current Genetics 50 (2): 81–99.
Charpentier, Emmanuelle, Hagen Richter, John van der Oost, and Malcolm F. White. 2015.
“Biogenesis Pathways of RNA Guides in Archaeal and Bacterial CRISPR-Cas Adaptive
Immunity.” FEMS Microbiology Reviews 39 (3): 428–41.
https://doi.org/10.1093/femsre/fuv023.
Chen, Fuqiang, Shondra M Pruett-Miller, Yuping Huang, Monika Gjoka, Katarzyna Duda, Jack
Taunton, Trevor N Collingwood, Morten Frodin, and Gregory D Davis. 2011. “High-
Frequency Genome Editing Using SsDNA Oligonucleotides with Zinc-Finger Nucleases.”
Nature Methods 8 (July): 753. https://doi.org/10.1038/nmeth.1653.
Christian, Michelle, Tomas Cermak, Erin L Doyle, Clarice Schmidt, Feng Zhang, Aaron Hummel,
Adam J Bogdanove, and Daniel F Voytas. 2010. “Targeting DNA Double-Strand Breaks
with TAL Effector Nucleases.” Genetics 186 (2): 757 LP-761.
https://doi.org/10.1534/genetics.110.120717.
“Clinical Trials Register.” n.d. Accessed January 21, 2019.
https://www.clinicaltrialsregister.eu/ctr-search/search.
Cong, Le, F Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D Hsu,
et al. 2013. “Multiplex Genome Engineering Using CRISPR/Cas Systems.” Science 339
(6121): 819 LP-823. https://doi.org/10.1126/science.1231143.
67
Cong, Le, Ruhong Zhou, Yu-chi Kuo, Margaret Cunniff, and Feng Zhang. 2012. “Comprehensive
Interrogation of Natural TALE DNA-Binding Modules and Transcriptional Repressor
Domains.” Nature Communications 3 (July): 968. https://doi.org/10.1038/ncomms1962.
Cyranoski, David. 2015. “Gene-Edited ‘micropigs’ to Be Sold as Pets at Chinese Institute.”
Nature 526 (7571): 18–18. https://doi.org/10.1038/nature.2015.18448.
———. 2016. “Chinese Scientists to Pioneer First Human CRISPR Trial.” Nature 535 (7613):
476–77. https://doi.org/10.1038/nature.2016.20302.
Dai, Wei-Jing, Li-Yao Zhu, Zhong-Yi Yan, Yong Xu, Qi-Long Wang, and Xiao-Jie Lu. 2016.
“CRISPR-Cas9 for in Vivo Gene Therapy: Promise and Hurdles.” Molecular Therapy -
Nucleic Acids 5 (January): e349. https://doi.org/10.1038/mtna.2016.58.
Dang, Yunkun, Qiuying Yang, Zhihong Xue, and Yi Liu. 2011. “RNA Interference in Fungi:
Pathways, Functions, and Applications.” Eukaryotic Cell 10 (9): 1148 LP-1155.
https://doi.org/10.1128/EC.05109-11.
Datsenko, Kirill A, Ksenia Pougach, Anton Tikhonov, Barry L Wanner, Konstantin Severinov,
and Ekaterina Semenova. 2012. “Molecular Memory of Prior Infections Activates the
CRISPR/Cas Adaptive Bacterial Immunity System.” Nature Communications 3 (July): 945.
https://doi.org/10.1038/ncomms1937.
Datta, Simanti, Nina Costantino, Xiaomei Zhou, and Donald L Court. 2008. “Identification and
Analysis of Recombineering Functions from Gram-Negative and Gram-Positive Bacteria
and Their Phages.” Proceedings of the National Academy of Sciences 105 (5): 1626 LP-
1631. https://doi.org/10.1073/pnas.0709089105.
Deglincerti, Alessia, Gist F Croft, Lauren N Pietila, Magdalena Zernicka-Goetz, Eric D Siggia,
and Ali H Brivanlou. 2016. “Self-Organization of the in Vitro Attached Human Embryo.”
Nature 533 (May): 251. https://doi.org/10.1038/nature17948.
Deltcheva, Elitza, Krzysztof Chylinski, Cynthia M Sharma, Karine Gonzales, Yanjie Chao, Zaid A
Pirzada, Maria R Eckert, Jörg Vogel, and Emmanuelle Charpentier. 2011. “CRISPR RNA
Maturation by Trans-Encoded Small RNA and Host Factor RNase III.” Nature 471 (March):
602. https://doi.org/10.1038/nature09886.
Deng, Ling, Roger A Garrett, Shiraz A Shah, Xu Peng, and Qunxin She. 2013. “A Novel
Interference Mechanism by a Type IIIB CRISPR-Cmr Module in Sulfolobus.” Molecular
Microbiology 87 (5): 1088–99. https://doi.org/10.1111/mmi.12152.
Dever, Daniel P, Rasmus O Bak, Andreas Reinisch, Joab Camarena, Gabriel Washington,
Carmencita E Nicolas, Mara Pavel-Dinu, et al. 2016. “CRISPR/Cas9 β-Globin Gene
Targeting in Human Haematopoietic Stem Cells.” Nature 539 (November): 384.
https://doi.org/10.1038/nature20134.
DiCarlo, James E, Alejandro Chavez, Sven L Dietz, Kevin M Esvelt, and George M Church. 2015.
“Safeguarding CRISPR-Cas9 Gene Drives in Yeast.” Nature Biotechnology 33 (November):
1250. https://doi.org/10.1038/nbt.3412.
Ding, Shou-Wei, and Olivier Voinnet. 2007. “Antiviral Immunity Directed by Small RNAs.” Cell
130 (3): 413–26. https://doi.org/10.1016/j.cell.2007.07.039.
68
Doench, John G. 2017. “Am I Ready for CRISPR? A User’s Guide to Genetic Screens.” Nature
Reviews Genetics 19 (2): 67–80. https://doi.org/10.1038/nrg.2017.97.
Doudna, Jennifer A., and Emmanuelle Charpentier. 2014. “The New Frontier of Genome
Engineering with CRISPR-Cas9.” Science 346 (6213): 1258096–1258096.
Doyon, Yannick, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jianbin Wang, Danny F
Xia, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory, and Michael C Holmes. 2010.
“Enhancing Zinc-Finger-Nuclease Activity with Improved Obligate Heterodimeric
Architectures.” Nature Methods 8 (December): 74.
https://doi.org/10.1038/nmeth.1539.
Dupuis, Marie-Ève, Manuela Villion, Alfonso H Magadán, and Sylvain Moineau. 2013. “CRISPR-
Cas and Restriction–modification Systems Are Compatible and Increase Phage
Resistance.” Nature Communications 4 (July): 2087.
https://doi.org/10.1038/ncomms3087.
Erdmann, Susanne, and Roger A Garrett. 2012. “Selective and Hyperactive Uptake of Foreign
DNA by Adaptive Immune Systems of an Archaeon via Two Distinct Mechanisms.”
Molecular Microbiology 85 (6): 1044–56. https://doi.org/10.1111/j.1365-
2958.2012.08171.x.
Esvelt, Kevin M., Prashant Mali, Jonathan L. Braff, Mark Moosburner, Stephanie J. Yaung, and
George M. Church. 2013. “Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation
and Editing.” Nature Methods 10 (11): 1116–21. https://doi.org/10.1038/nmeth.2681.
Esvelt, Kevin M, Andrea L Smidler, Flaminia Catteruccia, and George M Church. 2014.
“Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations.” Edited by
Diethard Tautz. ELife 3: e03401. https://doi.org/10.7554/eLife.03401.
Evers, Bastiaan, Katarzyna Jastrzebski, Jeroen P M Heijmans, Wipawadee Grernrum, Roderick
L Beijersbergen, and Rene Bernards. 2016. “CRISPR Knockout Screening Outperforms
ShRNA and CRISPRi in Identifying Essential Genes.” Nature Biotechnology 34 (April): 631.
https://doi.org/10.1038/nbt.3536.
Eyquem, Justin, Jorge Mansilla-Soto, Theodoros Giavridis, Sjoukje J C van der Stegen,
Mohamad Hamieh, Kristen M Cunanan, Ashlesha Odak, Mithat Gönen, and Michel
Sadelain. 2017. “Targeting a CAR to the TRAC Locus with CRISPR/Cas9 Enhances Tumour
Rejection.” Nature 543 (February): 113. https://doi.org/10.1038/nature21405.
“FDA Places CTX001 for SCD on Clinical Hold | MDedge Hematology and Oncology.” n.d.
Accessed January 21, 2019. https://www.mdedge.com/hematology-
oncology/article/184926/anemia/fda-places-ctx001-scd-clinical-hold.
Fineran, Peter C, and Emmanuelle Charpentier. 2012. “Memory of Viral Infections by CRISPR-
Cas Adaptive Immune Systems: Acquisition of New Information.” Virology 434 (2): 202–
9. https://doi.org/https://doi.org/10.1016/j.virol.2012.10.003.
Fineran, Peter C, Matthias J H Gerritzen, María Suárez-Diez, Tim Künne, Jos Boekhorst, Sacha
A F T van Hijum, Raymond H J Staals, and Stan J J Brouns. 2014. “Degenerate Target Sites
Mediate Rapid Primed CRISPR Adaptation.” Proceedings of the National Academy of
69
Sciences 111 (16): E1629 LP-E1638. https://doi.org/10.1073/pnas.1400071111.
Fonfara, Ines, Anaïs Le Rhun, Krzysztof Chylinski, Kira S Makarova, Anne-Laure Lécrivain, Janek
Bzdrenga, Eugene V Koonin, and Emmanuelle Charpentier. 2014. “Phylogeny of Cas9
Determines Functional Exchangeability of Dual-RNA and Cas9 among Orthologous Type
II CRISPR-Cas Systems.” Nucleic Acids Research 42 (4): 2577–90.
http://dx.doi.org/10.1093/nar/gkt1074.
Fulci, Valerio, and Giuseppe Macino. 2007. “Quelling: Post-Transcriptional Gene Silencing
Guided by Small RNAs in Neurospora Crassa.” Current Opinion in Microbiology 10 (2):
199–203. https://doi.org/https://doi.org/10.1016/j.mib.2007.03.016.
Gabriel, Richard, Angelo Lombardo, Anne Arens, Jeffrey C Miller, Pietro Genovese, Christine
Kaeppel, Ali Nowrouzi, et al. 2011. “An Unbiased Genome-Wide Analysis of Zinc-Finger
Nuclease Specificity.” Nature Biotechnology 29 (August): 816.
Gaj, Thomas, Jing Guo, Yoshio Kato, Shannon J Sirk, and Carlos F Barbas III. 2012. “Targeted
Gene Knockout by Direct Delivery of Zinc-Finger Nuclease Proteins.” Nature Methods 9
(July): 805. https://doi.org/10.1038/nmeth.2030.
Gantz, Valentino M, Nijole Jasinskiene, Olga Tatarenkova, Aniko Fazekas, Vanessa M Macias,
Ethan Bier, and Anthony A James. 2015. “Highly Efficient Cas9-Mediated Gene Drive for
Population Modification of the Malaria Vector Mosquito Anopheles Stephensi.”
Proceedings of the National Academy of Sciences 112 (49): E6736–43.
https://doi.org/10.1073/pnas.1521077112.
Garneau, Josiane E, Marie-Ève Dupuis, Manuela Villion, Dennis A Romero, Rodolphe
Barrangou, Patrick Boyaval, Christophe Fremaux, Philippe Horvath, Alfonso H Magadán,
and Sylvain Moineau. 2010. “The CRISPR/Cas Bacterial Immune System Cleaves
Bacteriophage and Plasmid DNA.” Nature 468 (November): 67.
Gaudelli, Nicole M, Alexis C Komor, Holly A Rees, Michael S Packer, Ahmed H Badran, David I
Bryson, and David R Liu. 2017. “Programmable Base Editing of T to G C in Genomic DNA
without DNA Cleavage.” Nature 551 (7681): 464–71.
———. 2018. “Publisher Correction: Programmable Base Editing of A•T to G•C in Genomic
DNA without DNA Cleavage.” Nature 551 (7714): 464–71.
https://doi.org/10.1038/s41586-018-0070-x.
Gent, Jonathan I, Ayelet T Lamm, Derek M Pavelec, Jay M Maniar, Poornima Parameswaran,
Li Tao, Scott Kennedy, and Andrew Z Fire. 2010. “Distinct Phases of SiRNA Synthesis in
an Endogenous RNAi Pathway in C. Elegans Soma.” Molecular Cell 37 (5): 679–89.
https://doi.org/10.1016/j.molcel.2010.01.012.
Ghildiyal, Megha, and Phillip D Zamore. 2009. “Small Silencing RNAs: An Expanding Universe.”
Nature Reviews Genetics 10 (February): 94. https://doi.org/10.1038/nrg2504.
Gilbert, Luke A., Matthew H. Larson, Leonardo Morsut, Zairan Liu, Gloria A. Brar, Sandra E.
Torres, Noam Stern-Ginossar, et al. 2013. “CRISPR-Mediated Modular RNA-Guided
70
Regulation of Transcription in Eukaryotes.” Cell 154 (2): 442–51.
https://doi.org/10.1016/j.cell.2013.06.044.
Goldberg, Gregory W, Wenyan Jiang, David Bikard, and Luciano A Marraffini. 2014.
“Conditional Tolerance of Temperate Phages via Transcription-Dependent CRISPR-Cas
Targeting.” Nature 514 (August): 633. https://doi.org/10.1038/nature13637.
Grissa, Ibtissem, Gilles Vergnaud, and Christine Pourcel. 2007. “CRISPRFinder: A Web Tool to
Identify Clustered Regularly Interspaced Short Palindromic Repeats.” Nucleic Acids
Research 35 (suppl_2): W52–57. http://dx.doi.org/10.1093/nar/gkm360.
Guilinger, John P, Vikram Pattanayak, Deepak Reyon, Shengdar Q Tsai, Jeffry D Sander, J Keith
Joung, and David R Liu. 2014. “Broad Specificity Profiling of TALENs Results in Engineered
Nucleases with Improved DNA-Cleavage Specificity.” Nature Methods 11 (February):
429. https://doi.org/10.1038/nmeth.2845.
Gupta, Ankit, Ryan G Christensen, Amy L Rayla, Abirami Lakshmanan, Gary D Stormo, and Scot
A Wolfe. 2012. “An Optimized Two-Finger Archive for ZFN-Mediated Gene Targeting.”
Nature Methods 9 (April): 588. https://doi.org/10.1038/nmeth.1994.
Haft, Daniel H, Jeremy Selengut, Emmanuel F Mongodin, and Karen E Nelson. 2005. “A Guild
of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist
in Prokaryotic Genomes.” PLOS Computational Biology 1 (6): e60.
https://doi.org/10.1371/journal.pcbi.0010060.
Hale, Caryn R., Sonali Majumdar, Joshua Elmore, Neil Pfister, Mark Compton, Sara Olson,
Alissa M. Resch, et al. 2012. “Essential Features and Rational Design of CRISPR RNAs That
Function with the Cas RAMP Module Complex to Cleave RNAs.” Molecular Cell 45 (3):
292–302. https://doi.org/https://doi.org/10.1016/j.molcel.2011.10.023.
Hale, Caryn R, Peng Zhao, Sara Olson, Michael O Duff, Brenton R Graveley, Lance Wells,
Rebecca M Terns, and Michael P Terns. 2009. “RNA-Guided RNA Cleavage by a CRISPR
RNA-Cas Protein Complex.” Cell 139 (5): 945–56.
https://doi.org/https://doi.org/10.1016/j.cell.2009.07.040.
Hammond, Andrew, Roberto Galizi, Kyros Kyrou, Alekos Simoni, Carla Siniscalchi, Dimitris
Katsanos, Matthew Gribble, et al. 2015. “A CRISPR-Cas9 Gene Drive System Targeting
Female Reproduction in the Malaria Mosquito Vector Anopheles Gambiae.” Nature
Biotechnology 34 (December): 78–83. https://doi.org/10.1038/nbt.3439.
Hammond, Scott M, Emily Bernstein, David Beach, and Gregory J Hannon. 2000. “An RNA-
Directed Nuclease Mediates Post-Transcriptional Gene Silencing in Drosophila Cells.”
Nature 404 (6775): 293–96. https://doi.org/10.1038/35005107.
Han, Zongchao, Shannon M Conley, Rasha S Makkia, Mark J Cooper, and Muna I Naash. 2012.
“DNA Nanoparticle-Mediated ABCA4 Delivery Rescues Stargardt Dystrophy in Mice.” The
Journal of Clinical Investigation 122 (9): 3221–26. https://doi.org/10.1172/JCI64833.
Hatoum-Aslan, Asma, Inbal Maniv, and Luciano A Marraffini. 2011. “Mature Clustered,
Regularly Interspaced, Short Palindromic Repeats RNA (CrRNA) Length Is Measured by a
Ruler Mechanism Anchored at the Precursor Processing Site.” Proceedings of the
National Academy of Sciences 108 (52): 21218 LP-21222.
71
Hatoum-Aslan, Asma, Poulami Samai, Inbal Maniv, Wenyan Jiang, and Luciano A Marraffini.
2013. “A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small
Interfering CRISPR RNAs.” The Journal of Biological Chemistry 288 (39): 27888–97.
https://doi.org/10.1074/jbc.M113.499244.
Heler, Robert, Poulami Samai, Joshua W Modell, Catherine Weiner, Gregory W Goldberg,
David Bikard, and Luciano A Marraffini. 2015. “Cas9 Specifies Functional Viral Targets
during CRISPR–Cas Adaptation.” Nature 519 (February): 199.
Hershlag, Avner, and Sara L Bristow. 2018. “Editing the Human Genome: Where ART and
Science Intersect.” Journal of Assisted Reproduction and Genetics 35 (8): 1367–70.
https://doi.org/10.1007/s10815-018-1219-0.
Hess, Gaelen T, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li, Karlene A Cimprich,
Stephen B Montgomery, and Michael C Bassik. 2016. “Directed Evolution Using DCas9-
Targeted Somatic Hypermutation in Mammalian Cells.” Nature Methods 13 (October):
1036. https://doi.org/10.1038/nmeth.4038.
Hille, Frank, Hagen Richter, Shi Pey Wong, Majda Bratovič, Sarah Ressel, and Emmanuelle
Charpentier. 2018. “The Biology of CRISPR-Cas: Backward and Forward.” Cell 172 (6):
1239–59. https://doi.org/10.1016/j.cell.2017.11.032.
Hochstrasser, Megan L., David W. Taylor, Jack E. Kornfeld, Eva Nogales, and Jennifer A.
Doudna. 2016. “DNA Targeting by a Minimal CRISPR RNA-Guided Cascade.” Molecular
Cell 63 (5): 840–51. https://doi.org/10.1016/J.MOLCEL.2016.07.027.
Hochstrasser, Megan L, David W Taylor, Prashant Bhat, Chantal K Guegler, Samuel H
Sternberg, Eva Nogales, and Jennifer A Doudna. 2014. “CasA Mediates Cas3-Catalyzed
Target Degradation during CRISPR RNA-Guided Interference.” Proceedings of the
Höck, Julia, and Gunter Meister. 2008. “The Argonaute Protein Family.” Genome Biology 9 (2):
210. https://doi.org/10.1186/gb-2008-9-2-210.
Hockemeyer, Dirk, Frank Soldner, Caroline Beard, Qing Gao, Maisam Mitalipova, Russell C
DeKelver, George E Katibah, et al. 2009. “Efficient Targeting of Expressed and Silent
Genes in Human ESCs and IPSCs Using Zinc-Finger Nucleases.” Nature Biotechnology 27
(August): 851. https://doi.org/10.1038/nbt.1562.
Hockemeyer, Dirk, Haoyi Wang, Samira Kiani, Christine S Lai, Qing Gao, John P Cassady,
Gregory J Cost, et al. 2011. “Genetic Engineering of Human Pluripotent Cells Using TALE
Nucleases.” Nature Biotechnology 29 (July): 731. https://doi.org/10.1038/nbt.1927.
Holkers, Maarten, Ignazio Maggio, Jin Liu, Josephine M Janssen, Francesca Miselli, Claudio
Mussolino, Alessandra Recchia, Toni Cathomen, and Manuel A F V Gonçalves. 2013.
“Differential Integrity of TALE Nuclease Genes Following Adenoviral and Lentiviral Vector
Gene Transfer into Human Cells .” Nucleic Acids Research 41 (5): e63–e63.
http://dx.doi.org/10.1093/nar/gks1446.
72
“Home - ClinicalTrials.Gov.” n.d. Accessed January 21, 2019.
https://clinicaltrials.gov/ct2/home.
“Home - Exonics Therapeutics.” n.d. Accessed January 21, 2019. http://exonicstx.com/.
“Home - Intellia Therapeutics.” n.d. Accessed January 21, 2019. https://www.intelliatx.com/.
“Home Page | CRISPR.” n.d. Accessed January 21, 2019. http://www.crisprtx.com/index.php.
Hou, Zhonggang, Yan Zhang, Nicholas E Propson, Sara E Howden, L.-F. Chu, Erik J Sontheimer,
and James A Thomson. 2013. “Efficient Genome Engineering in Human Pluripotent Stem
Cells Using Cas9 from Neisseria Meningitidis.” Proceedings of the National Academy of
Sciences 110 (39): 15644–49. https://doi.org/10.1073/pnas.1313587110.
Housden, Benjamin E, Alexander J Valvezan, Colleen Kelley, Richelle Sopko, Yanhui Hu, Charles
Roesel, Shuailiang Lin, et al. 2015. “Identification of Potential Drug Targets for Tuberous
Sclerosis Complex by Synthetic Screens Combining CRISPR-Based Knockouts with RNAi.”
Science Signaling 8 (393): rs9 LP-rs9. https://doi.org/10.1126/scisignal.aab3729.
Howes, Rob, and Christine Schofield. 2015. “Genome Engineering Using Adeno-Associated
Virus (AAV).” Methods in Molecular Biology (Clifton, N.J.) 1239: 75–103.
https://doi.org/10.1007/978-1-4939-1862-1_5.
Hsu, Patrick D., Eric S. Lander, and Feng Zhang. 2014. “Development and Applications of
CRISPR-Cas9 for Genome Engineering.” Cell 157 (6): 1262–78.
Hsu, Patrick D, David A Scott, Joshua A Weinstein, F Ann Ran, Silvana Konermann, Vineeta
Agarwala, Yinqing Li, et al. 2013. “DNA Targeting Specificity of RNA-Guided Cas9
Nucleases.” Nature Biotechnology 31 (9): 827–32. https://doi.org/10.1038/nbt.2647.
Huang, Chun-Hung, Claire R Shen, Hung Li, Li-Yu Sung, Meng-Ying Wu, and Yu-Chen Hu. 2016.
“CRISPR Interference (CRISPRi) for Gene Regulation and Succinate Production in
Cyanobacterium S. Elongatus PCC 7942.” Microbial Cell Factories 15 (1): 196.
https://doi.org/10.1186/s12934-016-0595-3.
Hutvagner, Gyorgy, and Martin J Simard. 2008. “Argonaute Proteins: Key Players in RNA
Silencing.” Nature Reviews Molecular Cell Biology 9 (January): 22.
https://doi.org/10.1038/nrm2321.
Hyun, Insoo, Amy Wilkerson, and Josephine Johnston. 2016. “Embryology Policy: Revisit the
14-Day Rule.” Nature 533 (7602): 169–71. https://doi.org/10.1038/533169a.
Isasi, R, E Kleiderman, and B M Knoppers. 2016. “Editing Policy to Fit the Genome?” Science
351 (6271): 337 LP-339. https://doi.org/10.1126/science.aad6778.
Ishino, Y, H Shinagawa, K Makino, M Amemura, and A Nakata. 1987. “Nucleotide Sequence of
the Iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia
Coli, and Identification of the Gene Product.” Journal of Bacteriology 169 (12): 5429 LP-
5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987.
Jackson, Ryan N., and Blake Wiedenheft. 2015. “A Conserved Structural Chassis for Mounting
Versatile CRISPR RNA-Guided Immune Responses.” Molecular Cell 58 (5): 722–28.
73
https://doi.org/10.1016/J.MOLCEL.2015.05.023.
Jackson, Ryan N, Sarah M Golden, Paul B G van Erp, Joshua Carter, Edze R Westra, Stan J J
Brouns, John van der Oost, Thomas C Terwilliger, Randy J Read, and Blake Wiedenheft.
2014. “Crystal Structure of the CRISPR RNA-Guided Surveillance Complex from
Escherichia Coli.” Science 345 (6203): 1473–79.
Jansen, Ruud., Jan. D A van Embden, Wim. Gaastra, and Leo. M Schouls. 2002. “Identification
of Genes That Are Associated with DNA Repeats in Prokaryotes.” Molecular Microbiology
43 (6): 1565–75. https://doi.org/10.1046/j.1365-2958.2002.02839.x.
Jeggo, P A. 1998. “DNA Breakage and Repair.” Advances in Genetics 38: 185–218.
Jiang, Wenyan, David Bikard, David Cox, Feng Zhang, and Luciano A Marraffini. 2013. “RNA-
Guided Editing of Bacterial Genomes Using CRISPR-Cas Systems.” Nature Biotechnology
31 (January): 233. https://doi.org/10.1038/nbt.2508.
Jiang, Yu, Biao Chen, Chunlan Duan, Bingbing Sun, Junjie Yang, and Sheng Yang. 2015.
“Multigene Editing in the Escherichia Coli Genome via the CRISPR-Cas9 System.” Edited
by R M Kelly. Applied and Environmental Microbiology 81 (7): 2506–14.
https://doi.org/10.1128/AEM.04023-14.
Jinek, Martin, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A Doudna, and
Emmanuelle Charpentier. 2012. “A Programmable Dual-RNA–Guided DNA Endonuclease
in Adaptive Bacterial Immunity.” Science 337 (6096): 816 LP-821.
Jinek, Martin, Fuguo Jiang, David W Taylor, Samuel H Sternberg, Emine Kaya, Enbo Ma, Carolin
Anders, et al. 2014. “Structures of Cas9 Endonucleases Reveal RNA-Mediated
Conformational Activation.” Science 343 (6176): 1247997.
Jore, Matthijs M, Magnus Lundgren, Esther van Duijn, Jelle B Bultema, Edze R Westra,
Sakharam P Waghmare, Blake Wiedenheft, et al. 2011. “Structural Basis for CRISPR RNA-
Guided DNA Recognition by Cascade.” Nature Structural &Amp; Molecular Biology 18
(April): 529. https://doi.org/10.1038/nsmb.2019.
Kabadi, Ami M, David G Ousterout, Isaac B Hilton, and Charles A Gersbach. 2014. “Multiplex
CRISPR/Cas9-Based Genome Engineering from a Single Lentiviral Vector.” Nucleic Acids
Research 42 (19): e147–e147. http://dx.doi.org/10.1093/nar/gku749.
Kang, Xiangjin, Wenyin He, Yuling Huang, Qian Yu, Yaoyong Chen, Xingcheng Gao, Xiaofang
Sun, and Yong Fan. 2016. “Introducing Precise Genetic Modifications into Human 3PN
Embryos by CRISPR/Cas-Mediated Genome Editing.” Journal of Assisted Reproduction
and Genetics 33 (5): 581–88. https://doi.org/10.1007/s10815-016-0710-8.
Karvelis, Tautvydas, Giedrius Gasiunas, Algirdas Miksys, Rodolphe Barrangou, Philippe
Horvath, and Virginijus Siksnys. 2013. “CrRNA and TracrRNA Guide Cas9-Mediated DNA
Interference in Streptococcus Thermophilus.” RNA Biology 10 (5): 841–51.
https://doi.org/10.4161/rna.24203.
Kiani, Samira, Alejandro Chavez, Marcelle Tuttle, Richard N Hall, Raj Chari, Dmitry Ter-
74
Ovanesyan, Jason Qian, et al. 2015. “Cas9 GRNA Engineering for Genome Editing,
Activation and Repression.” Nature Methods 12 (September): 1051.
Kim, Hye Joo, Hyung Joo Lee, Hyojin Kim, Seung Woo Cho, and Jin-Soo Kim. 2009. “Targeted
Genome Editing in Human Cells with Zinc Finger Nucleases Constructed via Modular
Assembly.” Genome Research 19 (7): 1279–88. https://doi.org/10.1101/gr.089417.108.
Kim, Yongsub, Jiyeon Kweon, Annie Kim, Jae Kyung Chon, Ji Yeon Yoo, Hye Joo Kim, Sojung
Kim, et al. 2013. “A Library of TAL Effector Nucleases Spanning the Human Genome.”
Nature Biotechnology 31 (February): 251. https://doi.org/10.1038/nbt.2517.
Klymiuk, Nikolai, Frank Seeliger, Mohammad Bohlooly-Y, Andreas Blutke, Daniel G Rudmann,
and Eckhard Wolf. 2016. “Tailored Pig Models for Preclinical Efficacy and Safety Testing
of Targeted Therapies.” Toxicologic Pathology 44 (3): 346–57.
https://doi.org/10.1177/0192623315609688.
Koike-Yusa, Hiroko, Yilong Li, E-Pien Tan, Martin Del Castillo Velasco-Herrera, and Kosuke
Yusa. 2013. “Genome-Wide Recessive Genetic Screening in Mammalian Cells with a
Lentiviral CRISPR-Guide RNA Library.” Nature Biotechnology 32 (December): 267.
Komor, Alexis C., Ahmed H. Badran, and David R. Liu. 2017. “CRISPR-Based Technologies for
the Manipulation of Eukaryotic Genomes.” Cell 168 (1): 20–36.
https://doi.org/10.1016/J.CELL.2016.10.044.
Komor, Alexis C, Yongjoo B Kim, Michael S Packer, John A Zuris, and David R Liu. 2016.
“Programmable Editing of a Target Base in Genomic DNA without Double-Stranded DNA
Cleavage.” Nature 533 (April): 420. https://doi.org/10.1038/nature17946.
Konermann, Silvana, Mark D Brigham, Alexandro E Trevino, Patrick D Hsu, Matthias
Heidenreich, Le Cong, Randall J Platt, David A Scott, George M Church, and Feng Zhang.
2013. “Optical Control of Mammalian Endogenous Transcription and Epigenetic States.”
Nature 500 (August): 472. https://doi.org/10.1038/nature12466.
Korkmaz, Gozde, Rui Lopes, Alejandro P Ugalde, Ekaterina Nevedomskaya, Ruiqi Han, Ksenia
Myacheva, Wilbert Zwart, Ran Elkon, and Reuven Agami. 2016. “Functional Genetic
Screens for Enhancer Elements in the Human Genome Using CRISPR-Cas9.” Nature
Biotechnology 34 (January): 192. https://doi.org/10.1038/nbt.3450.
Kraschel, Katherine L., and Natalie Kofler. 2018. “Introduction.” Seminars in Perinatology 42
(8): 485–86. https://doi.org/10.1053/j.semperi.2018.09.018.
Kuivanen, Joosu, Y.-M. Jasmin Wang, and Peter Richard. 2016. “Engineering Aspergillus Niger
for Galactaric Acid Production: Elimination of Galactaric Acid Catabolism by Using RNA
Sequencing and CRISPR/Cas9.” Microbial Cell Factories 15 (1): 210.
https://doi.org/10.1186/s12934-016-0613-5.
Kuscu, Cem, and Mazhar Adli. 2016. “CRISPR-Cas9-AID Base Editor Is a Powerful Gain-of-
Function Screening Tool.” Nature Methods 13 (November): 983.
Kuscu, Cem, Mahmut Parlak, Turan Tufan, Jiekun Yang, Karol Szlachta, Xiaolong Wei, Rashad
75
Mammadov, and Mazhar Adli. 2017. “CRISPR-STOP: Gene Silencing through Base-
Editing-Induced Nonsense Mutations.” Nature Methods 14 (June): 710.
Kuznedelov, Konstantin, Vladimir Mekler, Sofia Lemak, Monika Tokmina-Lukaszewska, Kirill
A. Datsenko, Ishita Jain, Ekaterina Savitskaya, et al. 2016. “Altered Stoichiometry
Escherichia Coli Cascade Complexes with Shortened CRISPR RNA Spacers Are Capable of
Interference and Primed Adaptation.” Nucleic Acids Research 44 (22): 10849–61.
https://doi.org/10.1093/nar/gkw914.
Lee, Young Je, Allison Hoynes-O’Connor, Matthew C Leong, and Tae Seok Moon. 2016.
“Programmable Control of Bacterial Gene Expression with the Combined CRISPR and
Antisense RNA System.” Nucleic Acids Research 44 (5): 2462–73.
http://dx.doi.org/10.1093/nar/gkw056.
Levy, Asaf, Moran G Goren, Ido Yosef, Oren Auster, Miriam Manor, Gil Amitai, Rotem Edgar,
Udi Qimron, and Rotem Sorek. 2015. “CRISPR Adaptation Biases Explain Preference for
Acquisition of Foreign DNA.” Nature 520 (April): 505.
Li, Guanglei, Yajing Liu, Yanting Zeng, Jianan Li, Lijie Wang, Guang Yang, Dunjin Chen, et al.
2017. “Highly Efficient and Precise Base Editing in Discarded Human Tripronuclear
Embryos.” Protein & Cell. Germany. https://doi.org/10.1007/s13238-017-0458-7.
Li, Ming, Rui Wang, Dahe Zhao, and Hua Xiang. 2014. “Adaptation of the Haloarcula Hispanica
CRISPR-Cas System to a Purified Virus Strictly Requires a Priming Process.” Nucleic Acids
Research 42 (4): 2483–92. http://dx.doi.org/10.1093/nar/gkt1154.
Li, Ting, Sheng Huang, Wen Zhi Jiang, David Wright, Martin H Spalding, Donald P Weeks, and
Bing Yang. 2011. “TAL Nucleases (TALNs): Hybrid Proteins Composed of TAL Effectors
and FokI DNA-Cleavage Domain.” Nucleic Acids Research 39 (1): 359–72.
http://dx.doi.org/10.1093/nar/gkq704.
Li, Zhongsen, Zhan-Bin Liu, Aiqiu Xing, Bryan P Moon, Jessica P Koellhoffer, Lingxia Huang, R
Timothy Ward, Elizabeth Clifton, S Carl Falco, and A Mark Cigan. 2015. “Cas9-Guide RNA
Directed Genome Editing in Soybean.” Plant Physiology 169 (2): 960 LP-970.
https://doi.org/10.1104/pp.15.00783.
Liang, Puping, Yanwen Xu, Xiya Zhang, Chenhui Ding, Rui Huang, Zhen Zhang, Jie Lv, et al.
2015. “CRISPR/Cas9-Mediated Gene Editing in Human Tripronuclear Zygotes.” Protein &
Cell 6 (5): 363–72. https://doi.org/10.1007/s13238-015-0153-5.
Lillestøl, Reidun K, Shiraz A Shah, Kim Brügger, Peter Redder, Hien Phan, Jan Christiansen, and
Roger A Garrett. 2009. “CRISPR Families of the Crenarchaeal Genus Sulfolobus:
Bidirectional Transcription and Dynamic Properties.” Molecular Microbiology 72 (1):
259–72. https://doi.org/10.1111/j.1365-2958.2009.06641.x.
Lino, Christopher A, Jason C Harper, James P Carney, and Jerilyn A Timlin. 2018. “Delivering
CRISPR: A Review of the Challenges and Approaches.” Drug Delivery 25 (1): 1234–57.
https://doi.org/10.1080/10717544.2018.1474964.
Liu, Tao, Yingjun Li, Xiaodi Wang, Qing Ye, Huan Li, Yunxiang Liang, Qunxin She, and Nan Peng.
76
2015. “Transcriptional Regulator-Mediated Activation of Adaptation Genes Triggers
CRISPR de Novo Spacer Acquisition.” Nucleic Acids Research 43 (2): 1044–55.
Long, Chengzu, Leonela Amoasii, Alex A Mireault, John R McAnally, Hui Li, Efrain Sanchez-
Ortiz, Samadrita Bhattacharyya, John M Shelton, Rhonda Bassel-Duby, and Eric N Olson.
2016. “Postnatal Genome Editing Partially Restores Dystrophin Expression in a Mouse
Model of Muscular Dystrophy.” Science 351 (6271): 400–403.
https://doi.org/10.1126/science.aad5725.
Lorenzo-Sellares, Víctor, Víctor Lorenzo, Armando Torres-Ramírez, Armando Torres, and
Eduardo Salido. 2014. “Primary Hyperoxaluria.” Nefrología (English Edition) 34 (3): 398–
412. https://doi.org/10.3265/Nefrologia.pre2014.Jan.12335.
Luo, Michelle L., Ryan N. Jackson, Steven R. Denny, Monika Tokmina-Lukaszewska, Kenneth
R. Maksimchuk, Wayne Lin, Brian Bothner, Blake Wiedenheft, and Chase L. Beisel. 2016.
“The CRISPR RNA-Guided Surveillance Complex in Escherichia Coli Accommodates
Extended RNA Spacers.” Nucleic Acids Research 44 (15): gkw421.
https://doi.org/10.1093/nar/gkw421.
Ma, Hong, Nuria Marti-Gutierrez, Sang-Wook Park, Jun Wu, Yeonmi Lee, Keiichiro Suzuki, Amy
Koski, et al. 2017. “Correction of a Pathogenic Gene Mutation in Human Embryos.”
Nature 548 (7668): 413–19. https://doi.org/10.1038/nature23305.
Ma, Yunqing, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, and Xing Chang. 2016.
“Targeted AID-Mediated Mutagenesis (TAM) Enables Efficient Genomic Diversification
in Mammalian Cells.” Nature Methods 13 (October): 1029.
Maeder, Morgan L, James F Angstman, Marcy E Richardson, Samantha J Linder, Vincent M
Cascio, Shengdar Q Tsai, Quan H Ho, et al. 2013. “Targeted DNA Demethylation and
Activation of Endogenous Genes Using Programmable TALE-TET1 Fusion Proteins.”
Nature Biotechnology 31 (October): 1137. https://doi.org/10.1038/nbt.2726.
Maeder, Morgan L, Samantha J Linder, Vincent M Cascio, Yanfang Fu, Quan H Ho, and J Keith
Joung. 2013. “CRISPR RNA–guided Activation of Endogenous Human Genes.” Nature
Methods 10 (July): 977. https://doi.org/10.1038/nmeth.2598.
Maeder, Morgan L, Stacey Thibodeau-Beganny, Anna Osiak, David A Wright, Reshma M
Anthony, Magdalena Eichtinger, Tao Jiang, et al. 2008. “Rapid ‘Open-Source’ Engineering
of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification.” Molecular
Cell 31 (2): 294–301. https://doi.org/10.1016/j.molcel.2008.06.016.
Maeder, Morgan L, Stacey Thibodeau-Beganny, Jeffry D Sander, Daniel F Voytas, and J Keith
Joung. 2009. “Oligomerized Pool Engineering (OPEN): An ‘open-Source’ Protocol for
Making Customized Zinc-Finger Arrays.” Nature Protocols 4 (10): 1471–1501.
https://doi.org/10.1038/nprot.2009.98.
Maier, Lisa-Katharina, Sita J Lange, Britta Stoll, Karina A Haas, Susan M Fischer, Eike Fischer,
Elke Duchardt-Ferner, Jens Wöhnert, Rolf Backofen, and Anita Marchfelder. 2013.
“Essential Requirements for the Detection and Degradation of Invaders by the Haloferax
Volcanii CRISPR/Cas System I-B.” RNA Biology 10 (5): 865–74.
77
https://doi.org/10.4161/rna.24282.
Makarova, Kira S., Daniel H. Haft, Rodolphe Barrangou, Stan J. J. Brouns, Emmanuelle
Charpentier, Philippe Horvath, Sylvain Moineau, et al. 2011. “Evolution and Classification
of the CRISPR–Cas Systems.” Nature Reviews Microbiology 9 (6): 467–77.
https://doi.org/10.1038/nrmicro2577.
Makarova, Kira S., Yuri I. Wolf, Omer S. Alkhnbashi, Fabrizio Costa, Shiraz A. Shah, Sita J.
Saunders, Rodolphe Barrangou, et al. 2015. “An Updated Evolutionary Classification of
CRISPR–Cas Systems.” Nature Reviews Microbiology 13 (11): 722–36.
https://doi.org/10.1038/nrmicro3569.
Makarova, Kira S, L Aravind, Yuri I Wolf, and Eugene V Koonin. 2011. “Unification of Cas
Protein Families and a Simple Scenario for the Origin and Evolution of CRISPR-Cas
Systems.” Biology Direct 6 (1): 38. https://doi.org/10.1186/1745-6150-6-38.
Makarova, Kira S, Nick V Grishin, Svetlana A Shabalina, Yuri I Wolf, and Eugene V Koonin. 2006.
“A Putative RNA-Interference-Based Immune System in Prokaryotes: Computational
Analysis of the Predicted Enzymatic Machinery, Functional Analogies with Eukaryotic
RNAi, and Hypothetical Mechanisms of Action.” Biology Direct 1 (1): 7.
https://doi.org/10.1186/1745-6150-1-7.
Makarova, Kira S, Yuri I Wolf, and Eugene V Koonin. 2013. “The Basic Building Blocks and
Evolution of CRISPR-CAS Systems.” Biochemical Society Transactions 41 (6): 1392–1400.
https://doi.org/10.1042/BST20130038.
Mali, Prashant, John Aach, P Benjamin Stranges, Kevin M Esvelt, Mark Moosburner, Sriram
Kosuri, Luhan Yang, and George M Church. 2013. “CAS9 Transcriptional Activators for
Target Specificity Screening and Paired Nickases for Cooperative Genome Engineering.”
Nature Biotechnology 31 (August): 833. https://doi.org/10.1038/nbt.2675.
Mali, Prashant, Luhan Yang, Kevin M Esvelt, John Aach, Marc Guell, James E DiCarlo, Julie E
Norville, and George M Church. 2013. “RNA-Guided Human Genome Engineering via
Cas9.” Science 339 (6121): 823 LP-826. https://doi.org/10.1126/science.1232033.
Mans, Robert, Harmen M van Rossum, Melanie Wijsman, Antoon Backx, Niels G A Kuijpers,
Marcel van den Broek, Pascale Daran-Lapujade, Jack T Pronk, Antonius J A van Maris, and
Jean-Marc G Daran. 2015. “CRISPR/Cas9: A Molecular Swiss Army Knife for Simultaneous
Introduction of Multiple Genetic Modifications in Saccharomyces Cerevisiae.” FEMS
Yeast Research 15 (2): fov004-fov004. http://dx.doi.org/10.1093/femsyr/fov004.
Marraffini, Luciano A., and Erik J. Sontheimer. 2010. “Self versus Non-Self Discrimination
during CRISPR RNA-Directed Immunity.” Nature 463 (7280): 568–71.
Marraffini, Luciano A, and Erik J Sontheimer. 2008. “CRISPR Interference Limits Horizontal
Gene Transfer in Staphylococci by Targeting DNA.” Science 322 (5909): 1843 LP-1845.
Meister, G, and T Tuschl. 2004. “Mechanisms of Gene Silencing by Double-Stranded RNA.”
Nature 431. https://doi.org/10.1038/nature02873.
Memi, Fani, Aglaia Ntokou, and Irinna Papangeli. 2018. “CRISPR/Cas9 Gene-Editing: Research
78
Technologies, Clinical Applications and Ethical Considerations.” Seminars in Perinatology
42 (8): 487–500. https://doi.org/https://doi.org/10.1053/j.semperi.2018.09.003.
Mendenhall, Eric M, Kaylyn E Williamson, Deepak Reyon, James Y Zou, Oren Ram, J Keith
Joung, and Bradley E Bernstein. 2013. “Locus-Specific Editing of Histone Modifications at
Endogenous Enhancers.” Nature Biotechnology 31 (September): 1133.
Miguel Beriain, Iñigo De, and Ana María Marcos del Cano. 2018. “Chapter 12 Gene Editing in
Human Embryos. A Comment on the Ethical Issues Involved.” In The Ethics of
Reproductive Genetics, edited by Marta (Ed.) Soniewicka, 1st ed., 173–87. Springer
International Publishing. https://doi.org/10.1007/978-3-319-60684-2_12.
Miller, Jeffrey C, Michael C Holmes, Jianbin Wang, Dmitry Y Guschin, Ya-Li Lee, Igor
Rupniewski, Christian M Beausejour, et al. 2007. “An Improved Zinc-Finger Nuclease
Architecture for Highly Specific Genome Editing.” Nature Biotechnology 25 (July): 778.
https://doi.org/10.1038/nbt1319.
Miller, Jeffrey C, Siyuan Tan, Guijuan Qiao, Kyle A Barlow, Jianbin Wang, Danny F Xia,
Xiangdong Meng, et al. 2010. “A TALE Nuclease Architecture for Efficient Genome
Editing.” Nature Biotechnology 29 (December): 143. https://doi.org/10.1038/nbt.1755.
Misra, Sanjukta. 2013. “Human Gene Therapy: A Brief Overview of the Genetic Revolution.”
The Journal of the Association of Physicians of India 61 (2): 127–33.
http://www.ncbi.nlm.nih.gov/pubmed/24471251.
Mohanraju, Prarthana, Kira S. Makarova, Bernd Zetsche, Feng Zhang, Eugene V. Koonin, and
John van der Oost. 2016. “Diverse Evolutionary Roots and Mechanistic Variations of the
CRISPR-Cas Systems.” Science 353 (6299): aad5147.
https://doi.org/10.1126/science.aad5147.
Mojica, F J M, C Díez-Villaseñor, J García-Martínez, and C Almendros. 2009. “Short Motif
Sequences Determine the Targets of the Prokaryotic CRISPR Defence System.”
Microbiology (Reading, England) 155 (Pt 3): 733–40.
https://doi.org/10.1099/mic.0.023960-0.
Mojica, Francisco J M, Cesar Diez-Villasenor, Jesus Garcia-Martinez, and Elena Soria. 2005.
“Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign
Genetic Elements.” Journal of Molecular Evolution 60 (2): 174–82.
https://doi.org/10.1007/s00239-004-0046-3.
Mollanoori, Hasan, and Shahram Teimourian. 2018. “Therapeutic Applications of CRISPR/Cas9
System in Gene Therapy.” Biotechnology Letters 40 (6): 907–14.
https://doi.org/10.1007/s10529-018-2555-y.
Morange, Michel. 2015. “What History Tells Us XXXVII. CRISPR-Cas: The Discovery of an
Immune System in Prokaryotes.” Journal of Biosciences 40 (2): 221–23.
https://doi.org/10.1007/s12038-015-9532-6.
Morbitzer, Robert, Patrick Römer, Jens Boch, and Thomas Lahaye. 2010. “Regulation of
Selected Genome Loci Using de Novo-Engineered Transcription Activator-like Effector
(TALE)-Type Transcription Factors.” Proceedings of the National Academy of Sciences 107
79
(50): 21617 LP-21622. https://doi.org/10.1073/pnas.1013133107.
Moscou, Matthew J, and Adam J Bogdanove. 2009. “A Simple Cipher Governs DNA
Recognition by TAL Effectors.” Science 326 (5959): 1501 LP-1501.
Mulepati, Sabin, Annie Héroux, and Scott Bailey. 2014. “Crystal Structure of a CRISPR RNA–
guided Surveillance Complex Bound to a SsDNA Target.” Science 345 (6203): 1479 LP-
1484. https://doi.org/10.1126/science.1256996.
Mulvihill, John J., Benjamin Capps, Yann Joly, Tamra Lysaght, Hub A. E. Zwart, and Ruth
Chadwick. 2017. “Ethical Issues of CRISPR Technology and Gene Editing through the Lens
of Solidarity.” British Medical Bulletin 122 (1): 17–29.
https://doi.org/10.1093/bmb/ldx002.
Musallam, Khaled M, Vijay G Sankaran, Maria Domenica Cappellini, Lorena Duca, David G
Nathan, and Ali T Taher. 2012. “Fetal Hemoglobin Levels and Morbidity in Untransfused
Patients with -Thalassemia Intermedia.” Blood 119 (2): 364–67.
https://doi.org/10.1182/blood-2011-09-382408.
Musallam, Khaled M, Ali T Taher, Maria Domenica Cappellini, and Vijay G Sankaran. 2013.
“Clinical Experience with Fetal Hemoglobin Induction Therapy in Patients with β-
Thalassemia.” Blood 121 (12): 2199 LP-2212. https://doi.org/10.1182/blood-2012-10-
408021.
Mussolino, Claudio, Robert Morbitzer, Fabienne Lütge, Nadine Dannemann, Thomas Lahaye,
and Toni Cathomen. 2011. “A Novel TALE Nuclease Scaffold Enables High Genome
Editing Activity in Combination with Low Toxicity.” Nucleic Acids Research 39 (21): 9283–
93. http://dx.doi.org/10.1093/nar/gkr597.
Nakayashiki, Hitoshi, Shugo Hanada, Nguyen Bao Quoc, Naoki Kadotani, Yukio Tosa, and
Shigeyuki Mayama. 2005. “RNA Silencing as a Tool for Exploring Gene Function in
Ascomycete Fungi.” Fungal Genetics and Biology 42 (4): 275–83.
https://doi.org/https://doi.org/10.1016/j.fgb.2005.01.002.
Nam, Ki Hyun, Charles Haitjema, Xueqi Liu, Fran Ding, Hongwei Wang, Matthew P. DeLisa, and
Ailong Ke. 2012. “Cas5d Protein Processes Pre-CrRNA and Assembles into a Cascade-like
Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System.” Structure 20 (9): 1574–
84. https://doi.org/https://doi.org/10.1016/j.str.2012.06.016.
Niewoehner, Jens, Bernd Bohrmann, Ludovic Collin, Eduard Urich, Hadassah Sade, Peter
Maier, Petra Rueger, et al. 2014. “Increased Brain Penetration and Potency of a
Therapeutic Antibody Using a Monovalent Molecular Shuttle.” Neuron 81 (1): 49–60.
https://doi.org/10.1016/j.neuron.2013.10.061.
Niewoehner, Ole, and Martin Jinek. 2016. “Structural Basis for the Endoribonuclease Activity
of the Type III-A CRISPR-Associated Protein Csm6.” RNA (New York, N.Y.) 22 (3): 318–29.
https://doi.org/10.1261/rna.054098.115.
Nishida, Keiji, Takayuki Arazoe, Nozomu Yachie, Satomi Banno, Mika Kakimoto, Mayura
Tabata, Masao Mochizuki, et al. 2016. “Targeted Nucleotide Editing Using Hybrid
Prokaryotic and Vertebrate Adaptive Immune Systems.” Science 353 (6305): aaf8729.
80
https://doi.org/10.1126/science.aaf8729.
Nishimasu, Hiroshi, F. Ann Ran, Patrick D. Hsu, Silvana Konermann, Soraya I. Shehata, Naoshi
Dohmae, Ryuichiro Ishitani, Feng Zhang, and Osamu Nureki. 2014. “Crystal Structure of
Cas9 in Complex with Guide RNA and Target DNA.” Cell 156 (5): 935–49.
Niu, Yuyu, Bin Shen, Yiqiang Cui, Yongchang Chen, Jianying Wang, Lei Wang, Yu Kang, et al.
2014. “Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene
Targeting in One-Cell Embryos.” Cell 156 (4): 836–43.
Nowak, Chance M, Seth Lawson, Megan Zerez, and Leonidas Bleris. 2016. “Guide RNA
Engineering for Versatile Cas9 Functionality.” Nucleic Acids Research 44 (20): 9555–64.
http://dx.doi.org/10.1093/nar/gkw908.
Nuñez, James K, Philip J Kranzusch, Jonas Noeske, Addison V Wright, Christopher W Davies,
and Jennifer A Doudna. 2014. “Cas1–Cas2 Complex Formation Mediates Spacer
Acquisition during CRISPR–Cas Adaptive Immunity.” Nature Structural &Amp; Molecular
Biology 21 (May): 528. https://doi.org/10.1038/nsmb.2820.
Nuñez, James K, Amy S Y Lee, Alan Engelman, and Jennifer A Doudna. 2015. “Integrase-
Mediated Spacer Acquisition during CRISPR–Cas Adaptive Immunity.” Nature 519
(February): 193. https://doi.org/10.1038/nature14237.
O’Connell, Mitchell R., Benjamin L. Oakes, Samuel H. Sternberg, Alexandra East-Seletsky,
Matias Kaplan, and Jennifer A. Doudna. 2014. “Programmable RNA Recognition and
Cleavage by CRISPR/Cas9.” Nature 516 (7530): 263–66.
Oh, Jee-Hwan, and Jan-Peter van Pijkeren. 2014. “CRISPR–Cas9-Assisted Recombineering in
Lactobacillus Reuteri.” Nucleic Acids Research 42 (17): e131–e131.
Oost, John van der, Edze R. Westra, Ryan N. Jackson, and Blake Wiedenheft. 2014.
“Unravelling the Structural and Mechanistic Basis of CRISPR–Cas Systems.” Nature
Reviews Microbiology 12 (7): 479–92. https://doi.org/10.1038/nrmicro3279.
ORBAN, TAMAS I, and ELISA IZAURRALDE. 2005. “Decay of MRNAs Targeted by RISC Requires
XRN1, the Ski Complex, and the Exosome.” RNA 11 (4): 459–69.
https://doi.org/10.1261/rna.7231505.
Palmer, Donna, and Philip Ng. 2003. “Improved System for Helper-Dependent Adenoviral
Vector Production.” Molecular Therapy 8 (5): 846–52.
https://doi.org/10.1016/j.ymthe.2003.08.014.
Palmer, Douglas C, Geoffrey C Guittard, Zulmarie Franco, Joseph G Crompton, Robert L Eil,
Shashank J Patel, Yun Ji, et al. 2015. “Cish Actively Silences TCR Signaling in CD8 + T Cells
to Maintain Tumor Tolerance.” The Journal of Experimental Medicine 212 (12): 2095–
2113. https://doi.org/10.1084/jem.20150304.
Parks, Robin J., Liane Chen, Martina Anton, Uma Sankar, Michael A. Rudnicki, and Frank L.
Graham. 1996. “A Helper-Dependent Adenovirus Vector System: Removal of Helper
81
Virus by Cre-Mediated Excision of the Viral Packaging Signal.” Proceedings of the
https://doi.org/10.1073/pnas.93.24.13565.
Parnas, Oren, Marko Jovanovic, Thomas M. Eisenhaure, Rebecca H. Herbst, Atray Dixit,
Chun Jimmie Ye, Dariusz Przybylski, et al. 2015. “A Genome-Wide CRISPR Screen in
Primary Immune Cells to Dissect Regulatory Networks.” Cell 162 (3): 675–86.
Pattanayak, Vikram, Steven Lin, John P Guilinger, Enbo Ma, Jennifer A Doudna, and David R
Liu. 2013. “High-Throughput Profiling of off-Target DNA Cleavage Reveals RNA-
Programmed Cas9 Nuclease Specificity.” Nature Biotechnology 31 (9): 839–43.
Pattanayak, Vikram, Cherie L Ramirez, J Keith Joung, and David R Liu. 2011. “Revealing Off-
Target Cleavage Specificities of Zinc-Finger Nucleases by in Vitro Selection.” Nature
Methods 8 (August): 765. https://doi.org/10.1038/nmeth.1670.
Pawluk, April, Nadia Amrani, Yan Zhang, Bianca Garcia, Yurima Hidalgo-Reyes, Jooyoung Lee,
Alireza Edraki, et al. 2016. “Naturally Occurring Off-Switches for CRISPR-Cas9.” Cell 167
(7): 1829–1838.e9. https://doi.org/10.1016/j.cell.2016.11.017.
Peng, Jin, Yong Wang, Junyi Jiang, Xiaoyang Zhou, Lei Song, Lulu Wang, Chen Ding, et al. 2015.
“Production of Human Albumin in Pigs Through CRISPR/Cas9-Mediated Knockin of
Human CDNA into Swine Albumin Locus in the Zygotes.” Scientific Reports 5 (November):
16705. https://doi.org/10.1038/srep16705.
Peng, Wenfang, Mingxia Feng, Xu Feng, Yun Xiang Liang, and Qunxin She. 2015. “An Archaeal
CRISPR Type III-B System Exhibiting Distinctive RNA Targeting Features and Mediating
Dual RNA and DNA Interference.” Nucleic Acids Research 43 (1): 406–17.
Pera, Martin F. 2017. “Human Embryo Research and the 14-Day Rule.” Development 144 (11):
1923 LP-1925. https://doi.org/10.1242/dev.151191.
Perez-Pinera, Pablo, D Dewran Kocak, Christopher M Vockley, Andrew F Adler, Ami M Kabadi,
Lauren R Polstein, Pratiksha I Thakore, et al. 2013. “RNA-Guided Gene Activation by
CRISPR-Cas9–based Transcription Factors.” Nature Methods 10 (July): 973.
Pijkeren, Jan Peter van, and Robert A Britton. 2014. “Precision Genome Engineering in Lactic
Acid Bacteria.” Microbial Cell Factories 13 (1): S10. https://doi.org/10.1186/1475-2859-
13-S1-S10.
Pingoud, Alfred, Geoffrey G. Wilson, and Wolfgang Wende. 2014. “Type II Restriction
Endonucleases—a Historical Perspective and More.” Nucleic Acids Research 42 (12):
7489–7527. https://doi.org/10.1093/nar/gku447.
Plagens, André, Vanessa Tripp, Michael Daume, Kundan Sharma, Andreas Klingl, Ajla Hrle,
Elena Conti, Henning Urlaub, and Lennart Randau. 2014. “In Vitro Assembly and Activity
of an Archaeal CRISPR-Cas Type I-A Cascade Interference Complex.” Nucleic Acids
Research 42 (8): 5125–38. http://dx.doi.org/10.1093/nar/gku120.
82
Porteus, Matthew H. 2017. “Genome Editing for the β-Hemoglobinopathies.” In Advances in
Experimental Medicine and Biology, 1013:203–17. United States.
https://doi.org/10.1007/978-1-4939-7299-9_8.
Pougach, Ksenia, Ekaterina Semenova, Ekaterina Bogdanova, Kirill A Datsenko, Marko
Djordjevic, Barry L Wanner, and Konstantin Severinov. 2010. “Transcription, Processing
and Function of CRISPR Cassettes in Escherichia Coli.” Molecular Microbiology 77 (6):
1367–79. https://doi.org/10.1111/j.1365-2958.2010.07265.x.
Pourcel, C., G. Salvignol, and Gilles Vergnaud. 2005. “CRISPR Elements in Yersinia Pestis
Acquire New Repeats by Preferential Uptake of Bacteriophage DNA, and Provide
Additional Tools for Evolutionary Studies.” Microbiology 151 (3): 653–63.
https://doi.org/10.1099/mic.0.27437-0.
Pul, Ümit, Reinhild Wurm, Zihni Arslan, René Geißen, Nina Hofmann, and Rolf Wagner. 2010.
“Identification and Characterization of E. Coli CRISPR-Cas Promoters and Their Silencing
by H-NS.” Molecular Microbiology 75 (6): 1495–1512. https://doi.org/10.1111/j.1365-
2958.2010.07073.x.
Pyne, Michael E, Murray Moo-Young, Duane A Chung, and C Perry Chou. 2015. “Coupling the
CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal
Gene Replacement in Escherichia Coli.” Edited by M Kivisaar. Applied and Environmental
Microbiology 81 (15): 5103–14. https://doi.org/10.1128/AEM.01248-15.
Qi, Lei S., Matthew H. Larson, Luke A. Gilbert, Jennifer A. Doudna, Jonathan S. Weissman,
Adam P. Arkin, and Wendell A. Lim. 2013. “Repurposing CRISPR as an RNA-Guided
Platform for Sequence-Specific Control of Gene Expression.” Cell 152 (5): 1173–83.
Qiu, Shibin, Coen M Adema, and Terran Lane. 2005. “A Computational Study of Off-Target
Effects of RNA Interference.” Nucleic Acids Research 33 (6): 1834–47.
http://dx.doi.org/10.1093/nar/gki324.
Radecke, Sarah, Frank Radecke, Toni Cathomen, and Klaus Schwarz. 2010. “Zinc-Finger
Nuclease-Induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted
Target Locus Modifications.” Molecular Therapy 18 (4): 743–53.
https://doi.org/10.1038/mt.2009.304.
Rajagopal, Nisha, Sharanya Srinivasan, Kameron Kooshesh, Yuchun Guo, Matthew D Edwards,
Budhaditya Banerjee, Tahin Syed, Bart J M Emons, David K Gifford, and Richard I
Sherwood. 2016. “High-Throughput Mapping of Regulatory DNA.” Nature Biotechnology
34 (January): 167. https://doi.org/10.1038/nbt.3468.
Ramia, Nancy F., Michael Spilman, Li Tang, Yaming Shao, Joshua Elmore, Caryn Hale, Alexis
Cocozaki, et al. 2014. “Essential Structural and Functional Roles of the Cmr4 Subunit in
RNA Cleavage by the Cmr CRISPR-Cas Complex.” Cell Reports 9 (5): 1610–17.
https://doi.org/https://doi.org/10.1016/j.celrep.2014.11.007.
Ramirez, Cherie L, Jonathan E Foley, David A Wright, Felix Müller-Lerch, Shamim H Rahman,
Tatjana I Cornu, Ronnie J Winfrey, et al. 2008. “Unexpected Failure Rates for Modular
Assembly of Engineered Zinc Fingers.” Nature Methods 5 (May): 374.
https://doi.org/10.1038/nmeth0508-374.
83
Ran, F. Ann, Patrick D. Hsu, Chie-Yu Lin, Jonathan S. Gootenberg, Silvana Konermann,
Alexandro E Trevino, David A. Scott, et al. 2013. “Double Nicking by RNA-Guided CRISPR
Cas9 for Enhanced Genome Editing Specificity.” Cell 154 (6): 1380–89.
Reyon, Deepak, Shengdar Q Tsai, Cyd Khayter, Jennifer A Foden, Jeffry D Sander, and J Keith
Joung. 2012. “FLASH Assembly of TALENs for High-Throughput Genome Editing.” Nature
Biotechnology 30 (April): 460. https://doi.org/10.1038/nbt.2170.
Richter, Corinna, Ron L Dy, Rebecca E McKenzie, Bridget N J Watson, Corinda Taylor, James T
Chang, Matthew B McNeil, Raymond H J Staals, and Peter C Fineran. 2014. “Priming in
the Type I-F CRISPR-Cas System Triggers Strand-Independent Spacer Acquisition, Bi-
Directionally from the Primed Protospacer.” Nucleic Acids Research 42 (13): 8516–26.
Ricroch, Agnès E, and Marie-Cécile Hénard-Damave. 2016. “Next Biotech Plants: New Traits,
Crops, Developers and Technologies for Addressing Global Challenges.” Critical Reviews
in Biotechnology 36 (4): 675–90. https://doi.org/10.3109/07388551.2015.1004521.
Robinson-Hamm, Jacqueline N, and Charles A Gersbach. 2016. “Gene Therapies That Restore
Dystrophin Expression for the Treatment of Duchenne Muscular Dystrophy.” Human
Genetics 135 (9): 1029–40. https://doi.org/10.1007/s00439-016-1725-z.
Rouet, P, F Smih, and M Jasin. 1994. “Expression of a Site-Specific Endonuclease Stimulates
Homologous Recombination in Mammalian Cells.” Proceedings of the National Academy
of Sciences 91 (13): 6064 LP-6068. https://doi.org/10.1073/pnas.91.13.6064.
Rouillon, Christophe, Min Zhou, Jing Zhang, Argyris Politis, Victoria Beilsten-Edmands,
Giuseppe Cannone, Shirley Graham, Carol V. Robinson, Laura Spagnolo, and Malcolm F.
White. 2013. “Structure of the CRISPR Interference Complex CSM Reveals Key Similarities
with Cascade.” Molecular Cell 52 (1): 124–34.
https://doi.org/https://doi.org/10.1016/j.molcel.2013.08.020.
Rudin, N., E. Sugarman, and J. E. Haber. 1989. “Genetic and Physical Analysis of Double-Strand
Break Repair and Recombination in Saccharomyces Cerevisiae.” Genetics 122 (3): 519–
34. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1203726/.
Ryan, Owen W, Jeffrey M Skerker, Matthew J Maurer, Xin Li, Jordan C Tsai, Snigdha Poddar,
Michael E Lee, et al. 2014. “Selection of Chromosomal DNA Libraries Using a Multiplex
CRISPR System.” Edited by Elisa Izaurralde. ELife 3: e03703.
https://doi.org/10.7554/eLife.03703.
Sampson, Timothy R, Sunil D Saroj, Anna C Llewellyn, Yih-Ling Tzeng, and David S Weiss. 2013.
“A CRISPR/Cas System Mediates Bacterial Innate Immune Evasion and Virulence.”
Nature 497 (April): 254. https://doi.org/10.1038/nature12048.
Sander, Jeffry D, Elizabeth J Dahlborg, Mathew J Goodwin, Lindsay Cade, Feng Zhang, Daniel
Cifuentes, Shaun J Curtin, et al. 2010. “Selection-Free Zinc-Finger-Nuclease Engineering
by Context-Dependent Assembly (CoDA).” Nature Methods 8 (December): 67.
Sapranauskas, Rimantas, Giedrius Gasiunas, Christophe Fremaux, Rodolphe Barrangou,
84
Philippe Horvath, and Virginijus Siksnys. 2011. “The Streptococcus Thermophilus
CRISPR/Cas System Provides Immunity in Escherichia Coli.” Nucleic Acids Research 39
(21): 9275–82. http://dx.doi.org/10.1093/nar/gkr606.
Sarkari, Parveen, Hans Marx, Marzena L Blumhoff, Diethard Mattanovich, Michael Sauer, and
Matthias G Steiger. 2017. “An Efficient Tool for Metabolic Pathway Construction and
Gene Integration for Aspergillus Niger.” Bioresource Technology 245: 1327–33.
https://doi.org/https://doi.org/10.1016/j.biortech.2017.05.004.
Sashital, Dipali G., Blake Wiedenheft, and Jennifer A. Doudna. 2012. “Mechanism of Foreign
DNA Selection in a Bacterial Adaptive Immune System.” Molecular Cell 46 (5): 606–15.
Sciences, National Academy of, National Academy of Medicine, and Engineering National
Academies of Sciences and Medicine. 2017. Human Genome Editing: Science, Ethics, and
Governance. Washington, DC: The National Academies Press.
https://doi.org/10.17226/24623.
Selker, E U, and J N Stevens. 1987. “Signal for DNA Methylation Associated with Tandem
Duplication in Neurospora Crassa.” Molecular and Cellular Biology 7 (3): 1032 LP-1038.
https://doi.org/10.1128/MCB.7.3.1032.
Selle, Kurt, and Rodolphe Barrangou. 2015a. “Harnessing CRISPR–Cas Systems for Bacterial
Genome Editing.” Trends in Microbiology 23 (4): 225–32.
https://doi.org/10.1016/j.tim.2015.01.008.
———. 2015b. “CRISPR-Based Technologies and the Future of Food Science.” Journal of Food
Science 80 (11): R2367–72. https://doi.org/10.1111/1750-3841.13094.
Selle, Kurt, Todd R Klaenhammer, and Rodolphe Barrangou. 2015. “CRISPR-Based Screening
of Genomic Island Excision Events in Bacteria.” Proceedings of the National Academy of
Sciences 112 (26): 8076 LP-8081. https://doi.org/10.1073/pnas.1508525112.
Semenova, Ekaterina, Matthijs M Jore, Kirill A Datsenko, Anna Semenova, Edze R Westra,
Barry Wanner, John van der Oost, Stan J J Brouns, and Konstantin Severinov. 2011.
“Interference by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNA
Is Governed by a Seed Sequence.” Proceedings of the National Academy of Sciences 108
(25): 10098 LP-10103. https://doi.org/10.1073/pnas.1104144108.
Shahbazi, Marta N, Agnieszka Jedrusik, Sanna Vuoristo, Gaelle Recher, Anna Hupalowska,
Virginia Bolton, Norah M E Fogarty, et al. 2016. “Self-Organization of the Human Embryo
in the Absence of Maternal Tissues.” Nature Cell Biology 18 (May): 700.
https://doi.org/10.1038/ncb3347.
Shalem, Ophir, Neville E Sanjana, Ella Hartenian, Xi Shi, David A Scott, Tarjei S Mikkelsen, Dirk
Heckl, et al. 2014. “Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.”
Science 343 (6166): 84 LP-87. https://doi.org/10.1126/science.1247005.
Sharpe, Pamela L, Deana DiCosimo, Melissa D Bosak, Kyle Knoke, Luan Tao, Qiong Cheng, and
Rick W Ye. 2007. “Use of Transposon Promoter-Probe Vectors in the Metabolic
Engineering of the Obligate Methanotroph Methylomonas Sp. Strain 16a for Enhanced
C40 Carotenoid Synthesis.” Applied and Environmental Microbiology 73 (6): 1721–28.
85
https://doi.org/10.1128/AEM.01332-06.
Shen, Binzhang, and Howard M Goodman. 2004. “Uridine Addition After MicroRNA-Directed
Cleavage.” Science 306 (5698): 997 LP-997. https://doi.org/10.1126/science.1103521.
Shipman, Seth L, Jeff Nivala, Jeffrey D Macklis, and George M Church. 2016. “Molecular
Recordings by Directed CRISPR Spacer Acquisition.” Science 353 (6298): aaf1175.
https://doi.org/10.1126/science.aaf1175.
Shiu, Patrick K T, Namboori B Raju, Denise Zickler, and Robert L Metzenberg. 2001. “Meiotic
Silencing by Unpaired DNA.” Cell 107 (7): 905–16. https://doi.org/10.1016/S0092-
8674(01)00609-2.
Smith, Cory, Athurva Gore, Wei Yan, Leire Abalde-Atristain, Zhe Li, Chaoxia He, Ying Wang, et
al. 2014. “Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9
and TALEN-Based Genome Editing in Human IPSCs.” Cell Stem Cell 15 (1): 12–13.
https://doi.org/10.1016/j.stem.2014.06.011.
Smithies, Oliver, Ronald G Gregg, Sallie S Boggs, Michael A Koralewski, and Raju S
Kucherlapati. 1985. “Insertion of DNA Sequences into the Human Chromosomal β-Globin
Locus by Homologous Recombination.” Nature 317 (September): 230.
https://doi.org/10.1038/317230a0.
Soldner, Frank, Josée Laganière, Albert W. Cheng, Dirk Hockemeyer, Qing Gao, Raaji
Alagappan, Vikram Khurana, et al. 2011. “Generation of Isogenic Pluripotent Stem Cells
Differing Exclusively at Two Early Onset Parkinson Point Mutations.” Cell 146 (2): 318–
31. https://doi.org/10.1016/j.cell.2011.06.019.
Spilman, Michael, Alexis Cocozaki, Caryn Hale, Yaming Shao, Nancy Ramia, Rebeca Terns,
Michael Terns, Hong Li, and Scott Stagg. 2013. “Structure of an RNA Silencing Complex
of the CRISPR-Cas Immune System.” Molecular Cell 52 (1): 146–52.
Staals, Raymond H.J., Yoshihiro Agari, Saori Maki-Yonekura, Yifan Zhu, David W. Taylor, Esther
van Duijn, Arjan Barendregt, et al. 2013. “Structure and Activity of the RNA-Targeting
Type III-B CRISPR-Cas Complex of Thermus Thermophilus.” Molecular Cell 52 (1): 135–
45. https://doi.org/https://doi.org/10.1016/j.molcel.2013.09.013.
Staals, Raymond H.J., Yifan Zhu, David W. Taylor, Jack E. Kornfeld, Kundan Sharma, Arjan
Barendregt, Jasper J. Koehorst, et al. 2014. “RNA Targeting by the Type III-A CRISPR-Cas
Csm Complex of Thermus Thermophilus.” Molecular Cell 56 (4): 518–30.
Standage-Beier, Kylie, Qi Zhang, and Xiao Wang. 2015. “Targeted Large-Scale Deletion of
Bacterial Genomes Using CRISPR-Nickases.” ACS Synthetic Biology 4 (11): 1217–25.
https://doi.org/10.1021/acssynbio.5b00132.
Sternberg, Samuel H, Sy Redding, Martin Jinek, Eric C Greene, and Jennifer A Doudna. 2014.
“DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9.” Nature 507
(January): 62. https://doi.org/10.1038/nature13011.
Streubel, Jana, Christina Blücher, Angelika Landgraf, and Jens Boch. 2012. “TAL Effector RVD
Specificities and Efficiencies.” Nature Biotechnology 30 (July): 593.
86
Suttle, Curtis A. 2005. “Viruses in the Sea.” Nature 437 (September): 356–61.
https://doi.org/https://doi.org/10.1038/nature04160.
Suzuki, Keiichiro, Chang Yu, Jing Qu, Mo Li, Xiaotian Yao, Tingting Yuan, April Goebl, et al.
2014. “Targeted Gene Correction Minimally Impacts Whole-Genome Mutational Load in
Human-Disease-Specific Induced Pluripotent Stem Cell Clones.” Cell Stem Cell 15 (1): 31–
36. https://doi.org/10.1016/j.stem.2014.06.016.
Svitashev, Sergei, Joshua K Young, Christine Schwartz, Huirong Gao, S Carl Falco, and A Mark
Cigan. 2015. “Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene
Insertion in Maize Using Cas9 and Guide RNA.” Plant Physiology 169 (2): 931 LP-945.
https://doi.org/10.1104/pp.15.00793.
Swarts, Daan C, Cas Mosterd, Mark W J van Passel, and Stan J J Brouns. 2012. “CRISPR
Interference Directs Strand Specific Spacer Acquisition.” PLOS ONE 7 (4): e35888.
https://doi.org/10.1371/journal.pone.0035888.
Sýkora, Peter. 2018. “Chapter 11 Germline Gene Therapy in the Era of Precise Genome
Editing: How Far Should We Go?” In The Ethics of Reproductive Genetics Between Utility,
Principles, and Virtues, edited by Marta Soniewicka, 157–71. Trnava: Springer
International Publishing AG. https://doi.org/10.1007/978-3-319-60684-2_11.
Szczepek, Michal, Vincent Brondani, Janine Büchel, Luis Serrano, David J Segal, and Toni
Cathomen. 2007. “Structure-Based Redesign of the Dimerization Interface Reduces the
Toxicity of Zinc-Finger Nucleases.” Nature Biotechnology 25 (July): 786.
https://doi.org/10.1038/nbt1317.
Tabara, Hiroaki, Madathia Sarkissian, William G Kelly, Jamie Fleenor, Alla Grishok, Lisa
Timmons, Andrew Fire, and Craig C Mello. 1999. “The Rde-1 Gene, RNA Interference, and
Transposon Silencing in C. Elegans.” Cell 99 (2): 123–32. https://doi.org/10.1016/S0092-
8674(00)81644-X.
Takeuchi, Nobuto, Yuri I. Wolf, Kira S. Makarova, and Eugene V. Koonin. 2012. “Nature and
Intensity of Selection Pressure on CRISPR-Associated Genes.” Journal of Bacteriology 194
(5): 1216–25. https://doi.org/10.1128/JB.06521-11.
Tamulaitis, Gintautas, Migle Kazlauskiene, Elena Manakova, Česlovas Venclovas, Alison O.
Nwokeoji, Mark J. Dickman, Philippe Horvath, and Virginijus Siksnys. 2014.
“Programmable RNA Shredding by the Type III-A CRISPR-Cas System of Streptococcus
Thermophilus.” Molecular Cell 56 (4): 506–17.
Tang, Lichun, Yanting Zeng, Hongzi Du, Mengmeng Gong, Jin Peng, Buxi Zhang, Ming Lei, et
al. 2017. “CRISPR/Cas9-Mediated Gene Editing in Human Zygotes Using Cas9 Protein.”
Molecular Genetics and Genomics : MGG 292 (3): 525–33.
https://doi.org/10.1007/s00438-017-1299-z.
“The CRISPR Economy: 7 Private Startups Pursuing The New Frontier In Biotech.” n.d.
Accessed January 21, 2019. https://www.cbinsights.com/research/crispr-startups-to-
watch/.
87
Thomas, K R, K R Folger, and M R Capecchi. 1986. “High Frequency Targeting of Genes to
Specific Sites in the Mammalian Genome.” Cell 44 (3): 419–28.
https://doi.org/10.1016/0092-8674(86)90463-0.
Thomas, Kirk R, and Mario R Capecchi. 1987. “Site-Directed Mutagenesis by Gene Targeting
in Mouse Embryo-Derived Stem Cells.” Cell 51 (3): 503–12.
https://doi.org/10.1016/0092-8674(87)90646-5.
Tomari, Yukihide, and Phillip D Zamore. 2005. “MicroRNA Biogenesis: Drosha Can’t Cut It
without a Partner.” Current Biology 15 (2): R61–64.
https://doi.org/10.1016/j.cub.2004.12.057.
Tong, Yaojun, Pep Charusanti, Lixin Zhang, Tilmann Weber, and Sang Yup Lee. 2015. “CRISPR-
Cas9 Based Engineering of Actinomycetal Genomes.” ACS Synthetic Biology 4 (9): 1020–
29. https://doi.org/10.1021/acssynbio.5b00038.
UNESCO. 1997. “OHCHR | Declaration on the Human Genome and Human Rights.” 1997.
https://www.ohchr.org/EN/ProfessionalInterest/Pages/HumanGenomeAndHumanRigh
ts.aspx.
Urnov, Fyodor D, Jeffrey C Miller, Ya-Li Lee, Christian M Beausejour, Jeremy M Rock, Sheldon
Augustus, Andrew C Jamieson, Matthew H Porteus, Philip D Gregory, and Michael C
Holmes. 2005. “Highly Efficient Endogenous Human Gene Correction Using Designed
Zinc-Finger Nucleases.” Nature 435 (April): 646. https://doi.org/10.1038/nature03556.
Urnov, Fyodor D, Edward J Rebar, Michael C Holmes, H Steve Zhang, and Philip D Gregory.
2010. “Genome Editing with Engineered Zinc Finger Nucleases.” Nature Reviews Genetics
11 (September): 636. https://doi.org/10.1038/nrg2842.
Wang, Haoyi, Hui Yang, Chikdu S Shivalila, Meelad M Dawlaty, Albert W Cheng, Feng Zhang,
and Rudolf Jaenisch. 2013. “One-Step Generation of Mice Carrying Mutations in Multiple
Genes by CRISPR/Cas-Mediated Genome Engineering.” Cell 153 (4): 910–18.
Wang, Lu, Zibo Zhao, Mark B. Meyer, Sandeep Saha, Menggang Yu, Ailan Guo, Kari B. Wisinski,
et al. 2014. “CARM1 Methylates Chromatin Remodeling Factor BAF155 to Enhance
Tumor Progression and Metastasis.” Cancer Cell 25 (1): 21–36.
https://doi.org/10.1016/j.ccr.2013.12.007.
Wang, Tim, Kıvanç Birsoy, Nicholas W Hughes, Kevin M Krupczak, Yorick Post, Jenny J Wei,
Eric S Lander, and David M Sabatini. 2015. “Identification and Characterization of
Essential Genes in the Human Genome.” Science 350 (6264): 1096 LP-1101.
https://doi.org/10.1126/science.aac7041.
Wang, Tim, Jenny J Wei, David M Sabatini, and Eric S Lander. 2014. “Genetic Screens in Human
Cells Using the CRISPR-Cas9 System.” Science 343 (6166): 80 LP-84.
“Weblet Importer.” n.d. Accessed January 20, 2019. https://www.editasmedicine.com/.
Wei, Yunzhou, Megan T Chesne, Rebecca M Terns, and Michael P Terns. 2015. “Sequences
Spanning the Leader-Repeat Junction Mediate CRISPR Adaptation to Phage in
Streptococcus Thermophilus.” Nucleic Acids Research 43 (3): 1749–58.
88
Wei, Yunzhou, Rebecca M Terns, and Michael P Terns. 2015. “Cas9 Function and Host Genome
Sampling in Type II-A CRISPR–Cas Adaptation.” Genes & Development 29 (4): 356–61.
https://doi.org/10.1101/gad.257550.114.
“Welcome to CGS | Center for Genetics and Society.” n.d. Accessed January 21, 2019.
https://www.geneticsandsociety.org/.
Westbrook, Adam W, Murray Moo-Young, and C Perry Chou. 2016. “Development of a
CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus Subtilis.” Edited by M
Kivisaar. Applied and Environmental Microbiology 82 (16): 4876–95.
https://doi.org/10.1128/AEM.01159-16.
Westra, Edze R., Paul B.G. van Erp, Tim Künne, Shi Pey Wong, Raymond H.J. Staals,
Christel L.C. Seegers, Sander Bollen, et al. 2012. “CRISPR Immunity Relies on the
Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade
and Cas3.” Molecular Cell 46 (5): 595–605.
Whitworth, Kristin M, Raymond R R Rowland, Catherine L Ewen, Benjamin R Trible, Maureen
A Kerrigan, Ada G Cino-Ozuna, Melissa S Samuel, et al. 2015. “Gene-Edited Pigs Are
Protected from Porcine Reproductive and Respiratory Syndrome Virus.” Nature
Biotechnology 34 (December): 20. https://doi.org/10.1038/nbt.3434.
WHO MEDIA CENTER. 2010. “WHO | Genes and Human Disease.” WHO. World Health
Organization. 2010.
https://www.who.int/genomics/public/geneticdiseases/en/index2.html#.XETrVRhG9e
w.mendeley.
Wiedenheft, Blake, Esther van Duijn, Jelle B Bultema, Sakharam P Waghmare, Kaihong Zhou,
Arjan Barendregt, Wiebke Westphal, et al. 2011. “RNA-Guided Complex from a Bacterial
Immune System Enhances Target Recognition through Seed Sequence Interactions.”
Proceedings of the National Academy of Sciences 108 (25): 10092 LP-10097.
Wiedenheft, Blake, Gabriel C Lander, Kaihong Zhou, Matthijs M Jore, Stan J J Brouns, John van
der Oost, Jennifer A Doudna, and Eva Nogales. 2011. “Structures of the RNA-Guided
Surveillance Complex from a Bacterial Immune System.” Nature 477 (September): 486.
Xie, Kabin, Bastian Minkenberg, and Yinong Yang. 2015. “Boosting CRISPR/Cas9 Multiplex
Editing Capability with the Endogenous TRNA-Processing System.” Proceedings of the
Xu, Chunlong, Xiaolan Qi, Xuguang Du, Huiying Zou, Fei Gao, Tao Feng, Hengxing Lu, et al.
2017. “PiggyBac Mediates Efficient in Vivo CRISPR Library Screening for Tumorigenesis in
Mice.” Proceedings of the National Academy of Sciences 114 (4): 722–27.
Xu, Tao, Yongchao Li, Zhou Shi, Christopher L Hemme, Yuan Li, Yonghua Zhu, Joy D Van
89
Nostrand, Zhili He, and Jizhong Zhou. 2015. “Efficient Genome Editing in Clostridium
Cellulolyticum via CRISPR-Cas9 Nickase.” Edited by A M Spormann. Applied and
Environmental Microbiology 81 (13): 4423–31. https://doi.org/10.1128/AEM.00873-15.
Yang, Luhan, Marc Guell, Susan Byrne, Joyce L Yang, Alejandro De Los Angeles, Prashant Mali,
John Aach, et al. 2013. “Optimization of Scarless Human Stem Cell Genome Editing.”
Nucleic Acids Research 41 (19): 9049–61. http://dx.doi.org/10.1093/nar/gkt555.
Yang, Luhan, Marc Güell, Dong Niu, Haydy George, Emal Lesha, Dennis Grishin, John Aach, et
al. 2015. “Genome-Wide Inactivation of Porcine Endogenous Retroviruses (PERVs).”
Science 350 (6264): 1101 LP-1104. https://doi.org/10.1126/science.aad1191.
Yosef, Ido, Moran G Goren, and Udi Qimron. 2012. “Proteins and DNA Elements Essential for
the CRISPR Adaptation Process in Escherichia Coli.” Nucleic Acids Research 40 (12): 5569–
76. http://dx.doi.org/10.1093/nar/gks216.
Zebec, Ziga, Andrea Manica, Jing Zhang, Malcolm F White, and Christa Schleper. 2014.
“CRISPR-Mediated Targeted MRNA Degradation in the Archaeon Sulfolobus
Solfataricus.” Nucleic Acids Research 42 (8): 5280–88.
Zetsche, Bernd, Jonathan S. Gootenberg, Omar O. Abudayyeh, Ian M. Slaymaker, Kira S.
Makarova, Patrick Essletzbichler, Sara E. Volz, et al. 2015. “Cpf1 Is a Single RNA-Guided
Endonuclease of a Class 2 CRISPR-Cas System.” Cell 163 (3): 759–71.
https://doi.org/10.1016/J.CELL.2015.09.038.
Zhan, Tianzuo, Niklas Rindtorff, Johannes Betge, Matthias P Ebert, and Michael Boutros. 2018.
“CRISPR/Cas9 for Cancer Research and Therapy.” Seminars in Cancer Biology, April.
https://doi.org/10.1016/j.semcancer.2018.04.001.
Zhang, Hong, Qiu-Xiang Cheng, Ai-Min Liu, Guo-Ping Zhao, and Jin Wang. 2017. “A Novel and
Efficient Method for Bacteria Genome Editing Employing Both CRISPR/Cas9 and an
Antibiotic Resistance Cassette.” Frontiers in Microbiology 8: 812.
https://doi.org/10.3389/fmicb.2017.00812.
Zhang, Jing, Christophe Rouillon, Melina Kerou, Judith Reeks, Kim Brugger, Shirley Graham,
Julia Reimann, et al. 2012. “Structure and Mechanism of the CMR Complex for CRISPR-
Mediated Antiviral Immunity.” Molecular Cell 45 (3): 303–13.
Zhang, Mingzi M, Fong Tian Wong, Yajie Wang, Shangwen Luo, Yee Hwee Lim, Elena Heng,
Wan Lin Yeo, et al. 2017. “CRISPR–Cas9 Strategy for Activation of Silent Streptomyces
Biosynthetic Gene Clusters.” Nature Chemical Biology 13 (April): 607.
https://doi.org/10.1038/nchembio.2341.
Zhang, Yan, Nadja Heidrich, Biju Joseph Ampattu, Carl W. Gunderson, H. Steven Seifert,
Christoph Schoen, Jörg Vogel, and Erik J. Sontheimer. 2013. “Processing-Independent
CRISPR RNAs Limit Natural Transformation in Neisseria Meningitidis.” Molecular Cell 50
(4): 488–503. https://doi.org/10.1016/j.molcel.2013.05.001.
Zhao, Hongtu, Gang Sheng, Jiuyu Wang, Min Wang, Gabor Bunkoczi, Weimin Gong, Zhiyi Wei,
and Yanli Wang. 2014. “Crystal Structure of the RNA-Guided Immune Surveillance
90
Cascade Complex in Escherichia Coli.” Nature 515 (7525): 147–50.
Zheng, Qiantao, Jun Lin, Jiaojiao Huang, Hongyong Zhang, Rui Zhang, Xueying Zhang, Chunwei
Cao, et al. 2017. “Reconstitution of UCP1 Using CRISPR/Cas9 in the White Adipose Tissue
of Pigs Decreases Fat Deposition and Improves Thermogenic Capacity.” Proceedings of
the National Academy of Sciences 114 (45): E9474–82.
Zhou, Changyang, Meiling Zhang, Yu Wei, Yidi Sun, Yun Sun, Hong Pan, Ning Yao, et al. 2017.
“Highly Efficient Base Editing in Human Tripronuclear Zygotes.” Protein & Cell. Germany.
https://doi.org/10.1007/s13238-017-0459-6.
Zhu, Xing, and Keqiong Ye. 2015. “Cmr4 Is the Slicer in the RNA-Targeting Cmr CRISPR
Complex.” Nucleic Acids Research 43 (2): 1257–67.
Zou, Qingjian, Xiaomin Wang, Yunzhong Liu, Zhen Ouyang, Haibin Long, Shu Wei, Jige Xin, et
al. 2015. “Generation of Gene-Target Dogs Using CRISPR/Cas9 System.” Journal of
Molecular Cell Biology 7 (6): 580–83. http://dx.doi.org/10.1093/jmcb/mjv061.
Zych, Agata O, Malgorzata Bajor, and Radoslaw Zagozdzon. 2018. “Application of Genome
Editing Techniques in Immunology.” Archivum Immunologiae et Therapiae
Experimentalis 66 (4): 289–98. https://doi.org/10.1007/s00005-018-0504-z.
91

CRISPR Technology Principles and Prospectives

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

CRISPR Technology Principles and Prospectives

Uploaded by

Copyright:

Available Formats

Zagazig University

CRISPR technology; principles

Mostafa Ahmed Mahmoud Elkhashab

Dr/ Ahmed Abdel-wahed Algazeery

I would like to express my special thanks of gratitude to my

There is nothing in this world that is quite as strong and enduring as a

2. Transcriptional Modulation ..................................................................... 43

FIG. 2. THE KEY STEPS OF CRISPR-CAS IMMUNITY ....................................................................... 9

FIG. 3. MODEL OF THE ADAPTATION......................................................................................... 11

FIG. 4. REPAIR OF DOUBLE-STRAND BREAKS ............................................................................. 23

FIG. 5. STRUCTURE OF ZFN........................................................................................................ 24

FIG. 6. STRUCTURE OF TALEN ................................................................................................... 25

FIG. 8. STRUCTURE OF CRISPR/CAS9 ......................................................................................... 31

FIG. 9. CRISPR-CAS9-MEDIATED GENOME EDITING................................................................... 32

FIG. 10. CAS9 CATALYTIC DOMAINS .......................................................................................... 33

FIG. 11. CAS9 NUCLEASE (NCAS9) ............................................................................................. 34

FIG. 12. HDR-MEDIATED TARGETED EDITING ............................................................................ 35

FIG. 13. CRISPR ACTIVATION ..................................................................................................... 36

FIG. 14. CRISPR INTERFERENCE ................................................................................................. 37

FIG. 15. SECOND-GENERATION CRISPR GENE EDITING TOOLS .................................................. 41

FIG. 16. TRANSCRIPTIONAL MODULATION ................................................................................ 43

FIG. 17. EPIGENETIC CONTROL .................................................................................................. 45

The immense intellectual energy surrounding CRISPR is evident in the speed

A Brief History of CRISPR Research from

A functional relationship for CRISPR/Cas was identified in 2005 by three

In 2009, it was discovered that CRISPR systems use a protospacer adjacent

CRISPR development was taken one step further by recreating a functional

Fig. 1. Timeline of CRISPR-Cas and genome engineering research fields.

When in vitro genetic engineering techniques using restriction endonucleases

The CRISPR-Cas immune system

Fig. 2. The key steps of CRISPR-Cas immunity. 1) Adaptation: insertion of new

Spacer selection appears guided by certain sequence elements in the target.

for spacer integration is conserved between the different systems remains to be

2. Expression of CRISPR RNA and cas genes

Diversity and classification of CRISPR-Cas

principal modules of the CRISPR-Cas systems consist of the suites of genes

The Class 1 systems

The Class 2 systems

Previous Genome editing tools and CRISPR

Zinc finger nucleases

Finally, a significant concern about the use of proteins designed to introduce

Transcription activator-like effector nucleases

In comparison with ZFNs, TALENs have turned out to be much easier to

A clear disadvantage of TALENs is their significantly larger size compared

miRNA is a common regulator to the endogenous genes, while siRNA is the

Fig. 8. Structure of CRISPR/Cas9. Cas9 recruitment to the target DNA is

Fig. 9. CRISPR-Cas9-mediated genome editing. DSB is repaired by Non-homologous end

Mechanisms of CRISPR-Cas9 tools

1. CRISPR-Cas9-mediated genome editing

One main application of CRISPR-Cas9 tools is DNA cleavage (Fig.12).

2. CRISPR-dCas9-mediated expression control

In addition to DNA cleavage, CRISPR-Cas9 tools exhibit functions of

Catalytically inactive Cas9 (dCas9) is fused with a transcription factor,

The dCas9 lacks endonuclease activity, however, when coexpressed with a

Advantages of CRISPR-Cas9 gene editing tool

Table 1. Comparison of CRISPR/Cas9, ZFNs, TALENs and RNAi

CRISPR/Cas9 ZFNs TALENs RNAi

Delivery systems for CRISPR

Viral Delivery Systems

Table 2. Summary of different delivery vectors.

Applications of CRISPR-Cas9 tools

Researchers recently harnessed the efficiency of this CRISPR base editor to

Cas9 can also be converted into a synthetic transcriptional activator by fusing