Received: 18 May 2018 | Accepted: 4 September 2018

DOI: 10.1002/jcp.27476

REVIEW ARTICLE

CRISPR–Cas9 in genome editing: Its function and medical

applications

Saedeh Khadempar1 | Shokoufeh Familghadakchi2 | Roozbeh Akbari Motlagh3 |

Najmeh Farahani 4
| Maryam Dashtiahangar 5
| Hamzeh Rezaei 6
|
7
Seyed Mohammad Gheibi Hayat

1
Departemant of Medical Genetics, Shahid
Sadoughi University of Medical Science, Abstract
Yazd, Iran The targeted genome modification using RNA‐guided nucleases is associated with
2
Department of Clinical Biochemistry, School
several advantages such as a rapid, easy, and efficient method that not only provides
of Paramedicine, Hamadan University of
Medical Sciences, Hamadan, Iran the manipulation and alteration of genes and functional studies for researchers, but
3
Department of Biochemistry and Molecular also increases their awareness of the molecular basis of the disease and development
Biology, Faculty of Veterinary, Shahid
Chamran University of Ahvaz, Ahvaz, Iran of new and targeted therapeutic approaches. Different techniques have been
4
Department of Genetics and Molecular emerged so far as the molecular scissors mediating targeted genome editing including
Biology, Isfahan University of Medical
zinc finger nuclease, transcription activator‐like effector nucleases, and clustered
Sciences, Isfahan, Iran
5
Department of Biology, Faculty of Sciences,
regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein
Ferdowsi University of Mashhad, 9 (Cas9). CRISPR–Cas9 is a bacterial immune system against viruses in which the
Mashhad, Iran
6
single‐strand RNA‐guided Cas9 nuclease is linked to the targeted complementary
Department of Clinical Biochemistry, School
of Medicine, Hamadan University of Medical sequences to apply changes. The advances made in the transfer, modification, and
Sciences, Hamadan, Iran emergence of specific solutions have led to the creation of different classes of
7
Department of Medical Biotechnology,
CRISPR–Cas9. Since this robust tool is capable of direct correction of disease‐causing
Faculty of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran mutations, its ability to treat genetic disorders has attracted the tremendous
attention of researchers. Considering the reported cases of nonspecific targeting of
Correspondence
Seyed Mohammad Gheibi Hayat, Department Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard,
of Medical Biotechnology, School of Medicine,
significant advances have been made in choosing guide RNA, new enzymes and
Mashhad University of Medical Sciences,
Mashhad 91779‐48564, Iran. methods for identifying misplaced targeting. Here, we highlighted the history and
Email: Gheibi65@yahoo.com
various direct aspects of CRISPR–Cas9, such as precision in genomic targeting,
system transfer and its control over correction events with its applications in future
biological studies, and modern treatment of diseases.

KEYWORDS
CRISPR–Cas9, genome editing, new treatments, specific targeting

1 | INTRODUCTION Before the development of technology, researchers used different

In recent years, the introduction of programmable nucleases
has greatly increased the efficiency of targeted genome editing.
The programmable nucleases can be classified into four main
classes.
CRISPR tools capable of breaking double‐stranded DNA to generate
variations in the genome. For example, DNA‐binding nucleases including
meganuclease (MN; Xu, Liu, & Pardinas, 2015), zinc finger nuclease
(ZFN; Jo, Kim, & Ramakrishna, 2015), transcription activator‐like
effector nucleases (TALEN; Scharenberg, Duchateau, & Smith, 2013),

J Cell Physiol. 2019;234:5751–5761. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 5751

5752 | KHADEMPAR
clustered regularly interspaced short palindromic repeats (CRISPR)–
CRISPR‐associated protein 9 (Cas9; Go & Stottmann, 2016) are the four
most recently discovered type types.
The function of all these tools is to identify and link specifically to the
target DNA and then create a double‐strand break (DSB). In the first
three classes, detecting DNA‐binding domain is derived from protein. As
a result, the use of these systems due to the need to design and engineer
the protein in any experiment is very difficult, time‐consuming, and costly.
The CRISPR–Cas system is able to detect target DNA‐binding domain
derived from RNA, so the system is very rapid, simple, and inexpensive.
The CRISPR–Cas is a bacterial adaptive immune system which first
discovered in 1987 (Lander, 2016).
Each of the above‐mentioned nucleases has been subject to
limitations. The MNs are similar to restriction enzymes and are
designed to be capable of targeting sequences of 14–40 bp. The MNs
have been used only for a short time due to deficiencies such as
inadequate specificity in detecting the target DNA and the need for
their restriction sites to integrate with the target sequence. ZFNs
and TALENs have relatively similar functions, although they differ in
DNA‐binding sites, and include trinucleotide and mononucleotide
patterns. These enzymes have separate DNA‐binding sites and a
nonspecific restriction site called FokI endonuclease. These char-
acteristics, however, have led to the superiority of these two
enzymes compared with MNs (Silva et al., 2011). The biggest
challenge for designing and manufacturing of ZFNs is the feature
of specific binding to the target. In the case of TALENs, in spite of
having a binding property, each of the TALE domains and each of the
target sequence bases require sophisticated molecular cloning
techniques to design the determined and conserved TALE compo-
nents. Finally, the advent of powerful CRISPR introduced far more
cost‐effective and easier methods for generating changes in the
genome and protein engineering compared with the earlier methods
(Figure 1; Joung & Sander, 2013).
Accurate and targeted editing capability at any spot in the
genome of beings for many years has been the aspiration of
scientists, and today scientists have come closer to this goal with
the discovery of CRISPR (Hsu, Lander, & Zhang, 2014).
Researchers are now able to knockout and knockin any gene
in the genome. The genome editing based on the CRISPR–Cas9
system is an efficient and high‐potential tool that can be
replaced by old methods such as ZFN and TALEN (Sander &
Joung, 2014).
2 | THE Cas9 AS A N I MMUNE SYSTEM IN
BACTERIA AND ARCHAEA
Ishino Y et al. (1987) identified CRISPR in Escherichia coli. They found that
these zones have a specific barcode called spacers with matches originate
from viral or plasmid genomes. The hypothesis of the involvement of
these repeats in the adaptive defense of prokaryotes was confirmed in
2007. In other words, the spacer sequence is formed in the bacterial
genome during exposure to phages, and the level of bacterial sensitivity
ET AL.
to be infections by phages depends on the contents of the spacer
sequence. Subsequent studies showed that the CRISPR works as a
complex with Cas9 endonuclease, so that the Cas9 protein‐encoding
gene is located in the vicinity of the CRISPR locus. The Cas9 creates a gap
in the target DNA or RNA sequences. The bacteria and archaea via
CRISPR–Cas9 protect their genome against the attack of the phage
nucleic acid and the integrating plasmids. In fact, CRISPR–Cas9 comes
with the help of the immune system and targets a number of invading
nucleic acids and proteins, including DNA and RNA, by coordinated
activity (Hale et al., 2009).
The invasive foreign DNA is broken down by the Cas nucleases,
and then part of it is placed in the CRISPR site between two repeated
sequences, in which case it is referred to as a spacer. The spacer
sequences are used as templates for the production of short CRISPR
RNAs (crRNAs) and form a complex with the transactivating crRNA
(tracrRNA) molecule (Brouns et al., 2008; Jinek et al., 2012).
These two sequences together, as a guide sequence, direct the
Cas9 protein towards the invasive DNA. Upon the Cas9 protein binds
to the invasive DNA, this protein cleaves the foreign DNA strand
complementary to the crRNA sequence and its opposite sequence
through the nuclease domains of NHN and RuvC1‐Like, respectively
(F. Jiang et al., 2016).
The CRISPR–Cas9 systems can be classified based on the
performance of their subunits into two main classes. The first class
of the CRISPR–Cas9 systems consisting of multisubunit effector RNA
complexes (types I, III, and IV), and the second CRISPR–Cas9 systems
are composed of single‐subunit RNA effector consisting of types II
and V (Figure 2; Barrangou et al., 2007).
3 | A TOOL FOR G ENOME EDITING
The modern targeted genome editing system using the CRISPR–Cas9
technology has two components: an endonuclease and guide RNA
with short sequence (Jinek et al., 2012). The targeted endonuclease is
a bacterial Cas9 enzyme derived from Streptococcus pyogenes. The
Cas9 nuclease has two DNA cleavage domains: HNH nuclease
domain and RuvC1‐like nuclease domain (F. Jiang et al., 2016),
leading to blunt DNA DSB (Figure 3).
The guide RNA (gRNA) in this system refers to the engineered
chimeric single‐strand RNA, which has both tracrRNA and bacterial
crRNA roles. The researcher designs 20‐bp 5′‐terminal nucleotides
related to the gRNA (homing device) against the gene sequence desired
for cleavage and editing. This 24‐nucleotide sequence guides the Cas9–
gRNA complex to the targeted gene location just upstream of the
protospacer adjacent motif (PAM) site via RNA–DNA binding. The PAM
sequence differs between the different bacterial strains and the various
CRISPR–Cas proteins, and this sequence is 5′‐NGG for S. pyogenes.
Therefore, the CRISPR–Cas system in S. pyogenes is directed to each
DNA sequence with 5′‐N20‐NGG and accurately blunt DNA DSB
(Garneau et al., 2010). The sequence detected by PAM occurring via
Cas9 enzyme is different based on the bacterial strains producing the
Cas9 nucleases. The Cas9 nuclease in S. pyogenes is a type II system and
KHADEMPAR ET AL. | 5753

F I G U R E 1 Insertion of foreign genomic segments in repeated sequences (a,b). crRNA: clustered regularly interspaced short palindromic
repeats RNA; tracrRNA: transactivating‐crRNA [Color figure can be viewed at wileyonlinelibrary.com]

most commonly used for genome editing (Makarova et al., 2015). It
should be noted that other commercial Cas9 enzymes detect other
PAM sequences (Ran, Hsu, Wright et al., 2013; Figure 4).
The DNA DSB repair occurs through two mechanisms which could be
observed in all types of organisms, called nonhomologous end‐joining
(NHEJ) and homology‐directed repair (HDR) pathways (Maruyama
et al., 2015).
The successful genome editing using CRISPR–Cas9 depends on the
gRNA and the PAM sequences, and only the target sequence that
immediately follows the PAM sequence is targeted for genome editing.
change the expression of a gene in the use of transcription factors.
The CRISPR system, which could target exclusively a specific site in
the genome, made it possible for researchers who direct the
transcription factors specifically towards a gene to alter the
expression of that gene. All of these methods are based on a general
principle, which is the link between the gene expression modifying
agent and the Cas9 passive protein. Because we tend to change only
the gene expression in this way, so we make mutation in the Cas9
protein to deactivate the nuclease domains, which is called dCas9. As
a result, the dead Cas9 (dCas9) is unable to cleave the DNA.

4 | REGULATION OF GE NE E X PRESSION 5 | DOW NREGU LA TION O F GENE

EXPRESSION
There are several ways to change the gene expression level, such as
epigenetic changes, transcription factors, and others. One of the main The first successful results of the gene expression downregulation
challenges of driving these factors specifically toward the gene is to using dCas9 were observed in E. coli. One of the easiest methods to
5754 | KHADEMPAR ET AL.

F I G U R E 2 Types and classes of CRISPR–Cas9 systems. CRISPR–Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐
associated protein 9; crRNA: CRISPR RNA; PAM: protospacer adjacent motif [Color figure can be viewed at wileyonlinelibrary.com]

(CXCR4), and tumor protein 53 (TP53). The method of binding these

transcriptional inhibitors is to link these agents to the protein
C‐terminal using the Cas9 protein engineering (Gilbert et al., 2013).
In another way to further reduce the gene expression, in addition to
the protein C‐terminal, a transcriptional inhibitor was attached to the
protein N‐terminal as well (Gilbert et al., 2014).

6 | UPREGULATION OF GENE EXPRESSION

In a simple method to increase the gene expression in E. coli, the ω

subunit of the RNA polymerase was linked to the dCas9 protein,
allowing the assemblage of the holoenzyme in the target promoter
F I G U R E 3 Nuclease domains of Cas9. Cas9: clustered regularly (Bikard et al., 2013). The gene expression in mammalian cells can be
interspaced short palindromic repeats‐associated protein 9; increased using VP64‐activation agents (a complex resulting from the
gRNA: guide RNA; PAM: protospacer adjacent motif [Color figure
binding of four VP16 proteins) and other transcription activators. To
can be viewed at wileyonlinelibrary.com]
further increase the expression level, the activator attaches to both
N‐ and C‐terminals of protein (Farzadfard, Perli, & Lu, 2013; Gao
turn the gene off is to select the gRNA so that dCas9 is located on
et al., 2014). In a sophisticated but efficient way to far more gene
the downstream of the transcription start site, thus preventing the
expression, they attached 10 peptides as epitopes, and then removed
transcription elongation step by the RNA polymerase (Bikard et al.,
2013; Larson et al., 2013). This kind of CRISPR system that causes the scFV domain of antibody specific for these epitopes and gave
them VP64 or other activators. Therefore, the decapeptide complex
the inhibition of gene expression is called CRISPR interference.
was attached to the C‐terminal of the dCas9 protein, which is called
Scientists were able to use dCas9 for reducing slightly the enhanced
SunTag array (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014).
green fluorescent protein (egfp) gene expression in the HEK293T cell
In another approach to activate transcription, the gRNA is
line. The use of dCas9 coupled with a gene expression inhibitor could
engineered instead of the dCas9 protein, and RNA molecules
increase the system efficiency to reduce gene expression. Therefore,
called aptamers binding to a protein instead of some of the loops.
they used the domains of transcriptional inhibitors such as the
This approach uses MS2 aptamer, such proteins that are bound
Krüptel‐associated box (or by binding four domains of mSin3 and
to this RNA are called MS2 coat proteins. In this method using
forming SID4X) to reduce by more than 80% the gene expression of
engineering, the MS2 coat protein is linked to transcription
the transferrin receptor CD71, C‐X‐C chemokine receptor type 4
activator or nonactivator (Konermann et al., 2015).
KHADEMPAR ET AL. | 5755

F I G U R E 4 Schema of CRISPR–Cas genome editing technology (a-g). CRISPR–Cas9: clustered regularly interspaced short palindromic
repeats (CRISPR)–CRISPR‐associated protein 9; dCas9: dead Cas9; DSB: double‐strand break; GFP: green fluorescent protein; HDR: homology‐
directed repair; NHEJ: nonhomologous end‐joining; PAM: protospacer adjacent motif; sgRNA: single guide RNA; ssRNA: single‐stranded RNA
[Color figure can be viewed at wileyonlinelibrary.com]

7 | APPROPRIATE DESIGN OF GRNA FOR
T H E CR I S P R –C a s 9 S Y S T E M
Recent advances in CRISPR–Cas9 in the in vitro, in vivo, and even in
animal models, such as rat, zebrafish, and monkeys, have led to its
widespread usage. It is noteworthy that, for other programmable
nucleases including ZFN, TALEN, and MN, the nonspecific nuclease
conduction to the target site on the genome is based on the DNA–
protein interaction.
In the CRISPR–Cas9 system, this happens based on the DNA binding
to the target RNA; therefore, this system can create a massive off‐target
cleavage sites in the genome (Zhang, Tee, Wang, Huang, & Yang, 2015).
In addition to the large amounts of off‐target cleavage sites on the
upstream sites of the PAM with ‐NGG sequences, the possibility of off‐
target cleavage sites has been observed on sites including the PAM
secondary sequence (NAG) for SpCas9 (Hsu et al., 2013). Consequently,
comprehensive studies have been done to design online databases to
identify potentially off‐target cleavage sites based on the sequence
similarity to the target site as well as mismatches along the crRNA
(Graham & Root, 2015). The proper gRNA design for the CRISPR–Cas
technique is one of the key steps to prevent off‐target cleavage sites.
Factors such as the specific position of nucleotides along gRNA,
surrounding site sequence, gRNA subsidence, guanine–cytosine (GC)
content along gRNA, and the appropriate secondary structure for gRNA
should be considered in design to achieve cleavage at the appropriate
site (Doench et al., 2016).
A study examined 218 different gRNAs at different sites of the
genome and monitored their activity, and determined that 3′ and also 20
(nucleotide adjacent to PAM) positions were clearly associated with
appropriate cleavage in 20‐nucleotide gRNA sequence (Doench et al.,
2014). In addition, structural studies have shown that the
20‐nucleotide segment plays an important role in the formation of the
5756 | KHADEMPAR
primary loop R, a triple‐stranded structure including gRNA hybrid with
target DNA strands when two DNA strands are broken up (Anders,
Niewoehner, Duerst, & Jinek, 2014). Adenine in this site has also been
reported to reduce cleavage rates by about 50%. In general, nucleotides
in the seed region are important in the proper cleavage efficiency
(Gagnon et al., 2014). Initially, it was believed that the nonseed sequence
was not decisive for the proper cleavage, but recent studies showed that
the presence of thymidine and guanine nucleotides at positions 2 and 3
would have a negative effect on the proper cleavage.
It has also been indicated that the adenine at position 6 of this
sequence plays an important role in the proper cleavage efficiency.
Generally speaking, the nucleotides at 2 and 4–6 positions in gRNA are
pivotal in binding to the target site, resulting in better recruitment of
Cas9 protein towards RNA/DNA heteroduplex. In the statistical study of
218 gRNAs, as mentioned earlier, it was observed that low or high GC
content had negative effects on proper cleavage and about 40–60% GC
content was needed for proper cleavage (Liu et al., 2016). The genomic
position of the gRNA binding to DNA also plays an important role in
cleavage, and it has been observed that the RNA binding to the proximal
promoter and the transcription onset are more efficient than in the
intergenic regions, since chromatin is more available in these regions
(Singh, Kuscu, Quinlan, Qi, & Adli, 2015). The formation of the secondary
structure by gRNA, especially in the seed sequence, is important to
recruit the Cas9 to the target site and also induces the active
endonuclease structure of this enzyme (Jinek et al., 2014).
One of the approaches proposed to reduce off‐target cleavage is the
use of truncated gRNA (tru‐gRNA), which has about 17–18 bases. The
tru‐gRNA produces less off‐target cleavage (about 500 times lower)
compared with the common 20‐nucleotide gRNA (Fu, Sander, Reyon,
Cascio, & Joung, 2014). This shortening should be made at the gRNA
5′‐terminal since shortening at the 3′‐terminal results in a decrease and
even loss of gRNA activity (Fu et al., 2013). Reducing more than three
nucleotides reduces or eliminates gRNA activity. It seems that the
common 20‐nucleotide gRNA generates more energy for Cas9, while
tru‐gRNA produces minimal energy for Cas9, thus preventing off‐target
cleavage. The gRNA with additional guanine at the 5′‐terminal
(5′‐GGX20 or GGGX 19‐5′) can be more specialized. Although there is
still no precise mechanism for this issue, the change in the gRNA
stability, its expression level or its secondary structure can be
considered (Kim et al., 2015). Some other factors that increase the
probability of a specific binding are the addition of the A‐U base pair to
the gRNA scaffold (the tracrRNA sequence) that stabilizes the scaffold
and the addition of the base pair to the hairpin loop in the spatial
structure of the scaffolding that increases the gRNA binding rate to the
target site and ultimately enhances the specific cleavage (Chen et al.,
2013). Figure 3 summarizes the stages of gRNA design.
8 | MORE EFFICIE NT A ND MORE
A C C U R A T E T Y P E S O F C R I S P R SY S T E M
Recent studies have shown that although every 20 nucleotides in the
gRNA sequence are necessary for specific function and binding to the
ET AL.
target, a few mismatches are tolerated in these locations, in turn
causing the off‐target cleavage in the genome (Cong et al., 2013; W.
Jiang, Bikard, Cox, Zhang, & Marraffini, 2013). The Cas9 nickase has
been introduced to prevent the double‐strand cleavage in nonspecific
site. As noted, the Cas9 protein has two cleavage domains, each one
creating cleavage in one of the DNA strands, so deactivating each of
these domains can provide an enzyme enabling single‐strand nick,
which is called nickase. In this case, the double‐strand cleavage needs
to be induced by two gRNAs, each linked to one of the strands, such
that the 5′‐terminal of these gRNAs is in the same direction and the
distance between these two 5′ offsets is 0–20 bp. In such a case, the
cleavage is created with an additional 5′‐terminal in the target DNA.
This system is known as double nicking. In this system, the nickase
with D10A mutation in the RuvC domain is used normally (Ran, Hsu,
Lin et al., 2013). The importance of using the nickase is that if any of
the gRNAs were nonspecifically linked to the site and created a
single‐strand nick, then the BEC (base excision repair) system would
repair it, and only the double‐strand cleavage created as a result of
binding two gRNAs in close proximity leads to the activation of NHEJ
or HDR pathways. It should be noted that no effective method has
yet been introduced to check the effects of a single‐strand nick. This
system significantly reduces the off‐target cleavage through the
performance of Cas9n is also slightly reduced compared to the wild
type of the Cas9 protein (Mali et al., 2013).
To improve the nickase system, the researchers introduced a more
precise type of Cas9, in which both cleavage domains were deactivated.
This type is called dCas9. Subsequently, the nonspecific Fok 1 cleavage
domain binding to the dCas9 N‐terminal develops a system called RNA‐
guided Fok 1 nuclease (RFN). In this system, the dCas9 acts as a carrier
and a director of Fok 1 to the cleavage position. The principle of the
system is similar to double nicking, with the difference that the Fok 1
nuclease activity requires dimerization with its counterpart in the
opposite strand; therefore, unlike the nickase system, the gRNA binding
in RFN alone will not lead to a single‐strand nick. Like double nicking,
the system also requires the design of two gRNAs, each one connected
to one of the DNA strands. In this case, 5′‐terminal of these gRNAs
should be toward each other and the effective distance between the
5′ gRNAs is bp (Tsai et al., 2014). In addition, a new system for
simultaneous expression of two or more gRNAs has been used to
optimize RFN as much as possible. In this system, the endoribonuclease
Csy4 is expressed along with the dCas9–FokI complex in the cell. The
cleavage site of this enzyme is also located on the upstream region of
the gRNA‐encoding sequence. In this way, the gRNAs are expressed as a
transcript under a single promoter, and then the Cys‐4 enzyme
separates them by creating cleavage at their own site.
In conventional gRNA expression systems, the guanine is
necessary to be designed before 5′ region of the gRNA because this
design is essential for the effective expression of RNA from the U6
promoter, whereas in the Cys‐4‐based expression model, 5′ region of
the transcript contains the first detection sequence of Cys‐4.
Therefore, the presence of guanine is not compulsory in the
5′ region of the gRNA. This issue is very important in the design of
gRNA because it broadens the range of gRNAs appropriate for the
KHADEMPAR ET AL.
target (Cheng et al., 2013; Guilinger, Thompson, & Liu, 2014). The
simultaneous use of tru‐gRNA and RFN in cancer cell lines and stem
cells provides a good cleavage at high levels (Wyvekens, Topkar,
Khayter, Joung, & Tsai, 2015). One of the major limitations of this
system is its large size that prevents it from being transmitted using
viral vectors (Tsai & Joung, 2016).
One of the important issues in the development of off‐target
cleavage in the genome is the prolonged expression of the
components in the CRISPR system, especially Cas9 in the cell,
because this raises the possibility of inducing off‐target cleavage.
The researchers assessed the crystalline structure of this
protein and explored a position for splitting the protein into two
fragments without affecting the cleavage domains. This new form,
called Split Cas9, has the capability of reassembling after exposure
to gRNA. However, it has been shown that the activity of this
enzyme is incompatible with the tru‐gRNA system (Wright et al.,
2015). Subsequently, to eliminate the dependence of the activity
of the Split Cas9 system on gRNA, dimerizable domains inducible
with photon or small molecules were used to induce dimerization
of this Cas9 types. Zetsche et al. applied the rapamycin‐dimerizing
domain. This domain makes it possible to assemble Cas9 in the
presence of rapamycin (Zetsche, Volz, Zhang, 2015). In another
study, Davis et al. inserted an intein inducible with 4‐hydroxyta-
moxifen (4‐HT) in the appropriate site in the Cas9 structure. This
intein only is excluded from the enzyme in the presence of 4‐HT.
As a result, the Cas9 enzyme is activated in the presence of
inducible compound and deactivated in the absence of the
compound. This increases the specific system up to 25% in human
cells (Davis, Pattanayak, Thompson, Zuris, & Liu, 2015). In
addition, a photoactivatable Cas9 has also recently been reported.
This type has been developed based on the adhesion of a
photoinducible dimerizing domains to the Split Cas9 subunits.
The obvious feature of this system is the reversibility that cannot
be found in rapamycin‐ or intein‐inducible systems (Nihongaki,
Kawano, Nakajima, & Sato, 2015).
Replacing the amino acid in the wild Cas9 sequence can increase
the binding power of this protein and thus cause intolerance to
mismatch in the gRNA sequence. The eSpCas9 is a variant of SpCas9
in which three alanine amino acids are replaced (A/K1003A/
R1060A848K). The tails are linked to the off‐target strand. This
replacement led to removal of off‐target cleavages in the genome and
also increased cleavage in the specific site compared with the wild
form (Slaymaker et al., 2016). In another study, the replacement of
four amino acids in the nonspecific binding site of the target DNA
strand with gRNA (N497A, R661A, Q695A, and Q926A) increased
binding energy, and so reduced significantly the off‐target cleavage in
the genome (Kleinstiver et al., 2015). Applying these two types of
enzymes with the same gRNA and in the same cell line will be very
significant to compare their function. The introduction of these two
new types has been a great help for more precise genome
engineering for the level of genetic disease treatment.
The discovery and introduction of other proteins with endonu-
clease activity used for genome engineering can provide more
| 5757
comprehensive insights for the effective application of this tool,
including Cpf1 protein whose function in human genome engineering
has recently been studied (Kim et al., 2016). The Cpf1 is another
popular variant of the Class II CRISPR system, named as the type V.
This endonuclease has structural and functional differences with
the Cas9:
1. Its activity is dependent only on the presence of a crRNA and
needs no tracrRNA,
2. Creation of a terminal cleavage that increases the insertion rate
of a gene cassette to the genome,
3. The PAM position is 5′‐TTTN‐3′, which is rich in thymidine,
4. Cpf1 only contains a cleavage domain as similar to RuvC (Zetsche,
Gootenberg et al., 2015).
Recently, it has been observed that this protein (Cpf1) has both
DNase and RNase properties, which cause the editing of the
precursor RNA to direct towards the target protein (Fonfara, Richter,
Bratovič, Le Rhun, & Charpentier, 2016). The crystalline structure of
this protein has also been well studied in the target site, which can
provide a comprehensive view of its functions (Kim et al.,
2016; Zetsche, Gootenberg et al., 2015).
9 | I N T R O D U C T I O N T O C R I S P R –C as 9
APP LICA TION S
The CRISPR–Cas9 method is often used for the dual inactivation of
the gene pair and detecting intergene correlation though this method
can be applied to investigate the response of cells in exposure to
drugs. The protein product of this correlation can be independently
investigated by protein assay techniques. Finally, the repair of this
correlation framework in eukaryotic cells can help understand the set
of messenger pathways involved in cancer, and thus how the
intergene correlation network affects tumorigenicity. In contrast,
the sensitivity level of recent treatments can be improved by using
lethal connections induced by new drugs. In this regard, the
important role of gRNA in this collection should be given special
attention and efforts should be made to improve this system by
promoting its design. In this way, it can increase the rate of specific
editions, reduce the number of false positive targets, and design the
gRNA that targets the functional protein domains. Since the
difference in the Cas9 expression between cells in a line and other
different lines affects the effectiveness of correlation and impair-
ment, several studies, especially on proteomics, are needed to
provide a more comprehensive map of this correlation framework. It
should be emphasized that this experimental work framework alone
is unusable to cancer cells, but it can be applied for studying complex
biosystems and diseases in a variety of eukaryotic cells with the
ability of lentiviral transduction and cultivation in the media (Canver
et al., 2017).
Clearly, all cancers are the result of numerous and diverse
mutations that lead to cell overproliferation and the appearance of
5758 | KHADEMPAR
malignant phenotypes. The event bed and the impaired domain of
these mutations can be classified into four distinct categories:
oncogenes, tumor suppressors, epigenetic agents, and chemotherapy
resistance‐inducing genes (Young et al., 2017).
The above characteristic has the ability to correct these
mutations and to treat partly their resulting cancer. Since the
oncogenetic changes in a number of cancers lead to increased cell
proliferation and malignancy, it is possible to target oncogenes, such
as the receptor tyrosine kinase ErbB2, directly using the CRISPR–
Cas9 method. In a point of view, the CRISPR–Cas9 method can
create carcinogenic mutations in human cell lines and animal models.
In this regard, the cell lines have been developed for lung cancers,
acute myeloid leukemia, liver cancer, and pancreatic cancer (Xue
et al., 2014). The CRISPR–Cas9 systems can also be used to
systematically examine the function of genes in human cells. The
lentiviral library of sgRNA can be used against the genes detected by
functional screening for sensitive analyzes of next‐generation
sequencing. The screening of this powerful library, with a loss of
function approach, is expected to facilitate the identification of genes
that play a major role in different bioprocesses, such as drug toxicity,
targeted therapeutic molecules, and the expression of specific
phenotypes (Zhou et al., 2014).
10 | SOME STUDIES CONDU CTE D USING
CRISPR –C a s 9 S Y S T E M
The screening for 2368 genes via the CRISPR–Cas9 system in
melanoma cells revealed a new targeted therapy for immunotherapy
of cancer using PD‐1 inhibitors.
In this study, the Cas9 protein was sustainably cloned initially
within cancer cells. Then, gRNA was designed for 2368 different
genes, each injected by a viral vector into its own cells. Eventually,
the modified cells were grafted into rats to evaluate the effect of PD‐
1 inhibitor.
A new targeted therapy called Ptpn2 was identified in addition
to the two mentioned types (PD‐L1 and CD47). The Ptpn2 gene
deletion increased the intensity of the immune signal pathway
that reduced tumor cell growth and ultimately cell death.
Subsequent studies should determine the mechanism of action
of Ptpn2 gene inhibitors in the treatment of cancer and whether
this gene can be used as an efficient novel targeted therapy
(Manguso et al., 2017).
This study attempted to reveal the mechanism of this step in
obtaining a new spacer sequence. At this stage, the Cas1–Cas2
proteins carry out the main role. Structural studies showed that, in
addition to these two proteins, a third factor called integration host
factor (IHF) is also involved in this mechanism. The IHF links to the
start site of a new spacer and bends DNA to a U‐shaped structure
that allows Cas1–Cas2 binding to both DNA segments. This finding
indicates that, unlike other Cas proteins, both Cas1 and Cas2
proteins detect their target often through the spatial shape and other
Cas proteins detect their sequences, such as PAM, at the target site.
ET AL.
It was previously shown that these two proteins are capable of saving
images and videos. Now, according to determine the mechanism of
action of Cas1–Cas2, there is the prospect that other information, in
addition to images and videos, can also be saved in CRISPR–Cas
systems (Wright et al., 2017).
The researchers used the CRISPR–Cas system and managed to
save pixel‐based information of black and white images and short
videos in the genome of a living bacterial cell population. In addition,
the findings of this study on the performance of Cas1 and Cas2
proteins provide very useful information from the adaptation stage
(Shipman, Nivala, Macklis, & Church, 2017).
Viral anti‐CRISPR proteins have been shown to improve the
function of the CRISPR–Cas system in genome editing. The use of
AcrIIA4 inhibitory protein in cells edited by the CRISPR–Cas system
has been about four times reduction in the off‐target occurrence rate
without affecting the on‐target performance of this system. Such
studies prove that the CRISPR–Cas editing system can be controlled
in the cell and body and can become the perfect method to engineer
the genome as accurately as possible (Shin et al., 2017).
11 | CRISPR –C as 9 T ECH NOLO GY FOR
IN HIBITING RETINAL A NGIOGEN ESIS
The vascular endothelial growth factor receptor 2 plays a major role
in the angiogenesis process. Using the CRISPR–Cas9 system and the
adeno‐associated virus (AAV) viral vector, researchers were able to
prevent the angiogenesis in the clinical model for the first time,
resulting in loss of vision. Further studies are needed to examine the
safety and efficacy of this strategy in the treatment of angiogenesis‐
related disorders (Huang et al., 2017). Scientists used CRISPR
technology to decode a rare and sometimes lethal childhood
syndrome (dyskeratosis congenita), which causes the gradual loss
of the ability to produce vital blood cells. Because the rat models of
telomere deficiency are unable to show the full effects of this
syndrome, the researchers through CRISPR technology managed to
induce two mutations associated with the disease to human stem
cells and observed that the cells developed short telomeres as the
same as the disease. The inhibition of downstream effects of this
deficiency showed no impact on the prevention of telomere short-
ening, but the gradual disappearance of the blood cells was treated
and the blood cells restarted to produce (Fok et al., 2017).
A study was conducted to investigate the correction of a
pathogenic gene mutation (MYBPC3) in a human embryo with
hypertrophic cardiomyopathy (a disease that ultimately causes heart
failure). In this study, it is interesting to note that the paternal
mutated gene cleaved by the CRISPR–Cas9 is more effective
replaced by maternal gene than synthesized through the DNA. The
result of human embryonic manipulation was much more efficient
than the manipulation of the induced pluripotent stem cells. The
efficiency was 72.2% in the human embryo and 17.61–27.9% in stem
cells, which indicates that the human embryo uses a DNA repair
mechanism different from the somatic and stem cells. The injection of
KHADEMPAR ET AL.
the Cas9 protein form for this test resulted in no off‐target in this
strategy (Ma et al., 2017).
In the treatment of many advanced cancers, the immunotherapy
can be very effective in some patients but ineffective in some other
patients. Researchers through the CRISPR–Cas9 system were able to
detect 100 essential genes in the cell line of the melanoma whose
presence is essential for the function of T cells. Deleting any of these
genes showed that the cancer cells had more resistance to T cell. This
study used the 2CT‐CRISPR technique in which T cells were
considered as an effector and melanoma cells as a target (Patel
et al., 2017).
Using the RNA‐targeting Cas9 (RCas9) technique, the research-
ers have been able to correct molecular mutations leading to
microsatellite repeat expansion diseases, such as myotonic dystro-
phies type 1 and type 2, hereditary common forms of ALS, and
Huntington’s disease.
In the RCas9 technique, instead of recruiting the engineered
Cas9 to DNA, it binds to the target RNA. The efficacy of this
technique is estimated at 95% or more in the treatment of
microsatellite repeat expansion diseases.
This study is important because of engineering a new form of
CRISPR–Cas9 system.
Moreover, this engineered system is easily presented to the
target tissue by a viral vector because of deleting the regions of the
engineered Cas9 protein sequence that is required to bind to DNA;
as a result, the size of this engineering form is smaller than its wild
type (Batra et al., 2017). Researchers used the CRISPR technology to
produce less fatty pigs that have thermogenic capacity. The pigs are
cold‐sensitive because of the absence of the UPC1 gene that is
responsible for regulating body temperature in other mammals,
especially when the air is cooled. Older pigs tolerate to some extent
the cold temperatures due to more fat in the body. The researchers
through the CRISPR–Cas9 system were able to link the rat UPC1
gene to the pig embryo. Grown pigs showed that they could
withstand cold temperatures and had about 24% less fat in their
body. After 6 months, the engineered pigs were killed to observe
other changes, but no abnormality was observed and it was reported
that all pigs had reproductive ability (Zheng et al., 2017). The target
gene in this study was superoxide dismutase 1, which became
knockout. The onset of the disease in the treated rat model was
postponed for 5 weeks, and the rat survived 1 month longer than
untreated rats. It cannot yet be said that the disease is treated, but
the system is necessary to be optimized to target a greater
percentage of cells (Gaj et al., 2017).
12 | IN VIVO AC TIVATION O F THE GEN E
E X P R E S S I O N B Y C R I S P R– C a s 9 S Y S T E M
In previous studies, dCas9 variant was always merged into an
activator domain such as VP64. The limitation of this system is the
large size of the chimeric sequence and it cannot be easily introduced
by AAV to the targeted cell or animal model.
| 5759
The new idea has been proposed in this study, according to
which:
1. Wild‐type Cas9 or dCas9 can be used to activate the target gene.
2. The Cas9 variant can be introduced to the system apart from the
switcher and gRNA in a separate AAV, which has no longer spatial
constraints for presentation (Liao et al., 2017).
13 | T H E W I L D ‐TYP E C as 9 TO AC TIVAT E
THE GENES
In this study (Liao et al., 2017), it has been shown that if the gRNA
sequence is 14–15 bp (known as dead gRNA or dgRNA), the Cas9 will
be directed to the target site but will be unable to cause the DSB.
In this study, the two MS2 domains (the SAM system) were used
to reassemble the components. The MS2 domains recruit the MPH
transcriptional activation complex. One of the advantages of this
approach is indirect actions of epigenetic changes by recruiting a
transcription machine, rather than being directly triggered by
recruiting the epigenetic modulators. The method presented in this
study has had promising results in the rat model for acute kidney
failure, type I diabetes, and muscular dystrophy (Liao et al., 2017).
14 | CO N CL US I O N
The discovery of CRISPR–Cas9 technology as a bacterial immune
system against pathogens and its usage as an efficient tool for making
targeted changes in the genome has led to a huge revolution in basic
biology research. The growing ability of this technology is indis-
putable in the systematic study of the gene function in mammalian
cells, genomic variations during the progression of cancers and other
diseases, and its potential for modifying genetic mutations causing
genetic disorders. In this regard, evidence suggests that future
studies have focused on optimizing this technology. Better under-
standing of the mechanisms of intracellular repair systems following
a DSB induced by Cas9 endonuclease improves the characteristics of
targeted genomic changes. The development of special methods is
required for the safe and efficient transfer of Cas9 protein and guide
RNA to cells and tissues for the proper utilization of this technology
in human gene therapy. Besides several advantages of CRISPR–Cas9
system, this system could be introduced to clinical applications in the
fast and easy way. For example, several studies indicated that
CRISPR–Cas9 system could be used as an effective approach for
creating a new generation of chimeric antigen receptor T cells.
However, there are still discussions about the safety and efficiency of
CRISPR–Cas9 technology. One of the biggest concerns on this
technology is the use of profitable individuals to make changes in
human embryos in line with eugenic goals. Hence, there is a need for
an accurate, responsible, and humanistic monitoring and control in
this area by an authoritative international organization (Krishan,
Kanchan, & Singh, 2016).
5760 | KHADEMPAR
OR CID
Seyed Mohammad Gheibi Hayat http://orcid.org/0000-0002-
1378-118X
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How to cite this article: Khadempar S, Familghadakchi S,
Motlagh RA, et al. CRISPR–Cas9 in genome editing: Its
function and medical applications. J Cell Physiol. 2019;234:
5751–5761. https://doi.org/10.1002/jcp.27476