Review
Genome-Editing Technologies: Concept, Pros, and
Cons of Various Genome-Editing Techniques and
Bioethical Concerns for Clinical Application
Sikandar Hayat Khan1
1Department of Pathology, PNS HAFEEZ Hospital, Pathology E-8, Islamabad, Islamabad 44400, Pakistan
The traditional healthcare system is at the doorstep for entering
into the arena of molecular medicine. The enormous knowl-
edge and ongoing research have now been able to demonstrate
methodologies that can alter DNA coding. The techniques used
to edit or change the genome evolved from the earlier attempts
like nuclease technologies, homing endonucleases, and certain
chemical methods. Molecular techniques like meganuclease,
transcription activator-like effector nucleases (TALENs), and
zinc-finger nucleases (ZFNs) initially emerged as genome-edit-
ing technologies. These initial technologies suffer from lower
specificity due to their off-targets side effects. Moreover, from
biotechnology’s perspective, the main obstacle was to develop
simple but effective delivery methods for host cell entry. Later,
small RNAs, including microRNA (miRNA) and small inter-
fering RNA (siRNA), have been widely adopted in the research
laboratories to replace lab animals and cell lines. The latest dis-
covery of CRISPR/Cas9 technology seems more encouraging by
providing better efficiency, feasibility, and multi-role clinical
application. This later biotechnology seem to take genome-
engineering techniques to the next level of molecular engineer-
ing. This review generally discusses the various gene-editing
technologies in terms of the mechanisms of action, advantages,
and side effects.
Over the last half century after post-DNA helical structure discovery,
the world has seen a continuous staircase outburst of various molec-
ular technologies, which are now heading forward toward transla-
tion into clinical and laboratory practice.1 Given the availability of
sequencing platforms, acquired wisdom about the micro-mechanics
at work within the genetic apparatus, and the introduction of user-
friendly nanotechnologies, it was possible for next-generation scien-
tists to manipulate the genetic codes at various levels.2 Over the last
two decades we saw a plethora of molecular techniques, which al-
lowed us to edit genes or their alter pathways, allowing humans for
the first time to micro-edit the DNA codes and further to alter the
mRNA fate through post-transcriptional modifications.3
Principally, genome-wide editing techniques can be interpreted as
methods where DNA sequences are changed by deletions, mRNA
processing, and post-transcriptional modifications to result in altered
gene expression, leading to functional behavior of proteins.4,5 Com-
326 Molecular Therapy: Nucleic Acids Vol. 16 June 2019 ª 2019 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
mon to these methods are three basic steps, including mechanisms
for genetic tool entry into the cell and later nucleus; altering gene tran-
scription and onward processing function; and, finally, the end-
output in the shape of a suppressed, overexpressed, or simply an
altered protein product.6,7 From a holistic point of view, the tech-
niques involve an apparently simplistic concept involving multiple re-
ceptor-ligand interactions; varying cell entry modes like lipofection,
sonification, and transfection; and further downstream pathway ef-
fects. Furthermore, these technologies are variable in terms of their
specificity and sensitivity, off-target effects, finances, and technique
expertise. The body’s immune response to accept the foreign genetic
elements within the cells can lead to the rejection of foreign tissues.
Moreover, molecular knowledge, in terms of methodology differences,
defining targetable diseases, innovative nanotechnology tools for gene
editing, and ethical aspects, also needs to be understood. The plat-
forms for these technologies are improving every day, with a plethora
of new data appearing due to technology miniaturization and automa-
tion and newer discoveries to improve the yield and specificity of
an edited product. Alongside the developmental improvement in
genome-wide engineering the regulatory work-up, standardization
protocols need to be devised to reduce inter and intra-method impre-
cision, defining the indications and contraindications of every tech-
nique to help improve the concept of personalized medicine.
This review briefly explains the available technologies, provides com-
parison and contrast between different genome-editing methods,
and identifies some newer versions of genome editing with possible
bioethical concerns.
Review Methodology
PubMed searches with the keywords genome-editing techniques or
gene-editing techniques in the last 10 and 5 years yielded a total of
4,466 and 4,054 references, except some historical and related refer-
ences. Specific searches for articles dealing with specific genome-edit-
ing methods included conventional genome-editing systems (n = 100),
https://doi.org/10.1016/j.omtn.2019.02.027.
Correspondence: Sikandar Hayat Khan, Department of Pathology, PNS HAFEEZ
Hospital, Pathology E-8, Islamabad, Islamabad 44400, Pakistan.
E-mail: sik_cpsp@yahoo.com
www.moleculartherapy.org
Review
Figure 1. A Consolidated Overview of Genome-Editing Techniques
chemical methods (n = 252), meganucleases (n = 83), zinc-finger
nucleases (ZFNs) (n = 890), transcription activator-like effector nucle-
ases (TALENs) (n = 1,136), homing endonucleases (n = 265), and
CRISPR (n = 11,421). The search was therefore limited to reviews
showing conceptual information of common techniques and compar-
ative information about ZFNs, TALENSs, and CRISPR technologies.
Finally, the literature was searched for learning newer and advanced
gene-editing methods and bioethical concerns associated with genome
biotechnologies.
Genome-Editing Techniques
The recent expansion and advancements in the field of biotechnology
provided us with information and insight into the biochemical and
molecular mechanisms to edit DNA and, thus, modify downstream
pathways. To date, multiple biotechnologies have shown promise
for clinical use, but the field of genome-editing technologies is rapidly
evolving and improving. The new techniques seem promising, but the
earlier ones have also been updated and improved. For simplicity and
consolidation, an overview of genome-editing techniques is presented
in Figure 1.
Representative genome-editing techniques are discussed below.
(1) Conventional genome-editing technique. In the true sense, the
technique may not relate with evolving genome-editing tech-
niques. As highlighted in Figure 1, it includes homologous
recombination related with gene intervention. While not much
in vogue or lab use today, the technique is based on physiological
processes involving a double-stranded repair system. However,
some recent data have shown RAD52 protein to be important
in mediating homologous recombination, and this protein there-
fore has been considered as a therapy target in certain cancers like
BRCA 1 and 2 repair pathways.6,7 However, the technique as of
now could not gain widespread introduction due to the emer-
gence of newer techniques.
(2) Chemical modalities of genome editing. Komiyama8 utilized
non-restriction enzyme methodology termed artificial restriction
DNA cutter (ARCUT). This method uses pseudo-complemen-
tary peptide nucleic acid (pcPNA), whose job is to specify the
cleavage site within the chromosome or the telomeric region.
Once pcPNA specifies the site, excision here is carried out by
cerium (CE) and EDTA (chemical mixture), which performs
the splicing function.8 Furthermore, the technology uses a
DNA ligase that can later attach any desirable DNA within the
spliced site. The advantage of this particular technique is that it
can be used in high salt concentrations. Upon initial introduc-
tion, the technique looked quite appealing to the clinical market;
however, later issues like increased turnaround time and specif-
ically the manufacturing of site-specific pcPNA became huge
hurdles (Figure 2).8,9
(3) Homing endonuclease systems. Homing endocucleases (HEs)
with this word “homing” practically is interpreted as lateral trans-
mission of a genome DNA sequence. The general concept involves
a DNA segment where a site is removed by the endocnulceases,
Molecular Therapy: Nucleic Acids Vol. 16 June 2019 327
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Figure 2. Excision of Selective Site of dsDNA by Utilizing Artificial Restriction DNA Cutter
which thus results in the formation of 2 segments of DNA frag-
ment.10 So what are these HEs? They are nucleases that occur
naturally, with a size almost equivalent to 14 bp, and they are
capable of splicing slightly larger DNA sequences.11 Recently,
the introduction of recombinant adeno-associated viruses
(rAAVs) have allowed them as efficient vehicles for transporting
genetic tools of genome engineering into the cell, as depicted in
Figure 3.12 Issues pertaining to this technology include engineer-
ing difficulties in the preparation of these nucleases as well as
developing vectors for their entry into cells.13 Another issue
with rAAV, though improving with better biotechnology, was
328 Molecular Therapy: Nucleic Acids Vol. 16 June 2019
off-target effects like reducing site specificity, less DNA integra-
tion, and possible host genome mutations.14
(4) Protein-based nuclease systems. These systems incorporate
nuclease proteins for DNA sequence editing. The common tech-
niques are described below.
Meganucleases. Also termed molecular DNA scissors, these are
large base pair structures that are sometimes found in the genome.
Their potential to excise large pieces of DNA sequences was
recently recognized as a genetic tool to modify DNA. This genetic
potential has been manipulated in labs by modifying the recogni-
tion sites to create nicks, as required for DNA sequence change.
Figure 3. Schematic Showing rAAV Entry,
Movement within Cytoplasm, Attachment with
DNA, and Integration with DNA Segment for
Possible Genome Modification
The steps include the following: (1) entry of rAAV into cell,
(2) uptake by exosome and transport within cytoplasm, (3)
release of rAAV for entry into nucleus, (4) rAAV delivery of
homing endocnulease (HE) and desirable DNA segment,
(5) HE cut of the non-desirable DNA code, and (6) rAAV-
delivered desirable DNA code replacement of the DNA.
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Figure 4. Schematic Showing Step by Step Zinc-Finger Nuclease-Induced Genome Editing
The mechanisms include the following: (1) ZFNs containing FokI endonucleases and protein-binding domains are introduced into the cell, (2) FokI and protein-binding
domains are released to enter the nucleus, (3) protein-binding domains attach with DNA fragment to be removed, (3) FokI cuts out the identified DNA segment by creating
double-stranded DNA break, and (4) the desirable DNA segment is inserted and integrated into the DNA sequence.
These meganucleases are sometimes joined by proteins to create
large variants like DmoCre and E-Drel, which can further provide
nucleotide site-specific cleavage.15 The technique revolves around
two basic steps: first is the recognition of a cleavage site, and then
endonucleases splice out the region.16 The positive aspect related
to meganucleases is less toxicity, as they are naturally occurring
and provide very specific site cleavage. However, there are newer
techniques now in the clinical arena that have not allowed them to
flourish more.
ZFNs. ZFNs are purely artificial structures generated by a combi-
natorial approach where restriction endonucleases are joined with
zinc-finger-binding domain protein. Figure 4 explains their mech-
anism of action in detail, where a binding protein domain iden-
tifies after reaching the desirable splice site, which is then cut at
a specific codon by special restriction endonucleases called FokI.
The biotechnology is restricted in terms of attachment with 3 co-
dons on either side of the DNA chain. The technique in recent
years has gained widespread popularity due to its simplicity and
specificity, and it is being employed in clinical usage for certain
diseases.17,18
TALENs. TALENS almost resemble ZFNs in terms of
manufacturing and mode of action. They are made by a similar
principle where a restriction nuclease is bound to a DNA-binding
protein domain called TAL effector.19 The difference between
TALENs and ZFNs is that the former can target 3 nt in one go
and the latter can only address 1 nt, thus making TALENs slightly
more site specific with fewer off-target effects.20 However, the
techniques share many similarities (Figure 5).
(5) RNA DNA systems. These systems primarily include the
different types of CRISPR methods. The concept of CRISPR is
primitive and has been derived from an ancient immunity sys-
tem, adopted in nature by some prokaryotic cells like Archea
and probably some bacteria.21 CRISPR in itself has two compo-
nents, including SPR termed sometimes as spacers, which are
hallmarked by varying and differing nucleotide sequences, and
probably each one of them represents a past exposure to foreign
antigen. The CRI may represent the genetic memory for a bacte-
rium and can be re-activated once encountered with a similar
foreign antigen. CRI has similar nucleotides (repeats) represent-
ing like separators between different CRIs.22 Figure 6 attempts to
provide a basic overview of the CRISPR/Cas9 concept.
Cas especially Cas9 as depicted in Figure 6 has a nuclease function.
Whenever CRISPR RNA (crRNA; also termed guide RNA
[gRNA]) guides the Cas9 protein regarding a possible antigenic
threat, like a bacteriophage, it with the help of gRNA creates dou-
ble-stranded DNA (dsDNA) nicks at the guided selected sites,
causing a site-specific cleavage and, thus, destruction of the anti-
gen.23 Moreover, the memory from the antigen is stored as spacer
within CRISPR.24
This physiological role of Cas9/CRISPR as explained above had
recently been extensively utilized for multiple clinical condi-
tions.25–27 At the time of writing this review, the news broke about
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Figure 5. Diagram Showing Mechanisms of Transcription Activator-like Effector Nucleases

The steps of gene editing include the following: (1) TALENs containing FokI endonucleases and TALE domains are introduced into the cell, (2) FokI and TALE domains are
released to enter the nucleus, (3) TALE recognizes the non-desirable DNA segments and attaches with them, (4) FokI cleaves the non-desirable DNA segments, and (5) after
the non-desirable DNA segments are cleaved, the desirable segment of DNA is incorporated into the DNA.

Lulu and Nana being claimed to be the first genetically modified
babies, where the human genome was edited to create resistance
against HIV infection.28 Specific crRNA/gRNA has been engi-
neered, which can be introduced into cell nuclei and later Cas9
where the non-desirable dsDNA is associated with the Cas9 after
guidance provided by the specific crRNA/gRNA. This comple-
mentary binding between gRNA and the non-desirable segment
allows Cas9 to destroy the DNA fragment. In clinical and research
practice, the created nick can be specifically filled by inserting the
sequence of choice to change the non-desirable sequence of nucle-
otides.29
Over the last few years, CRISPR/Cas9 technology has gained wide-
spread popularity on account of its simplicity and specificity, with
different versions of the original now under research. Multiple ex-
perimentations and biotechnologies have been re-defining the
CRISPR/Cas technologies into 3 distinct types of CRISPR-Cas
types, based on crRNA processing and further action, including
the following:
Type 1 CRISPR/Cas system. This version utilized Cas5 or Cas6 for
pre-processing of crRNA; further cleavage function needs Cas3,
Cascade, and crRNA for interference.
Type 2 CRISPR/Cas system. Though Cas9 typically functions un-
der the guidance of crRNA to target DNA, RNase III, trans acti-
vating RNA (tracrRNA), and a yet-to-be-identified protein factor
are involved in trimming at the 50 end.
Type 3 CRISPR/Cas system. Like the type 1 system, this category
uses Cas6 for processing crRNA 30 end trimming. The uniqueness
of this technique is its targeting of RNA, which is done by a specific
complex called type III Csm/Cmr complex.30
Apart from the aforementioned conventional style classification of
CRISPR/Cas classification, the data review provided multiple
other biotechnologies now being utilized. Some examples include
photo-activating CRISPR system,31 Intein-inducible split Cas9,32
and modifications like hybrid crRNA-tracrRNA.33
(6) Gene-silencing techniques. These methods may not fall truly under
genome editing, but they still are capable of modifying the DNA
sequence. These technologies include RNAi, CRISPR interference
(CRISPRi), and morpholino oligonucleotide techniques.34–36
Comparative Analysis
The above provides a gist of the various commonly used genome-edit-
ing techniques. Though there is enormous development, innovation,
and design of newer ways to edit the genome, we focus our further dis-
cussion on the comparison of common techniques, including ZFN,
TALEN, and CRISPR methods. Tables 1, 2, and 3 provide a compara-
tive assessment among these methods.
Advancements in Genome Engineering
The biotechnology is booming with a lot of newer modalities to edit
the genome. Oligodeoxyribonucleotide (ODN) can be utilized with

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Figure 6. Schematic Demonstrating the Concept of CRISPR/Cas9 Interactions Leading to the Destruction of Viral Genome at the Selected Splice Site by the
crRNA/gRNA

double-stranded transcription factor decoy (TFD) to act as a thera-
peutic target for multiple diseases, which can affect the transcription
factor and thus bring in the requisite change in transcription and
further downstream protein actions.37 Papaioannou et al.38 have uti-
lized single-stranded ODNs to precisely cut genomes for repairing
very small point mutations, giving a footprint-free genome-editing
modality. This concept involves a drug (doxycycline)-induced Cs9
transgene, which is carried into the cell by a specific transposon,
providing us with very specific and efficient Cas9-mediated editing
of the genome. This technique does not need the conventional donor
template, and, thus, it is termed footprint-free genome editing.38 The
technique seems to have minimal off-target effects and is considered
to be a safer version.
Other novel modalities of genome editing are also appearing in the
literature, with slight modifications of existing techniques. Martínez-
Gálvez et al.39 used single-stranded DNA (ssDNA) and argonautes
in gene editing and helped improved gene editing. Some researchers
have utilized certain enzymes like integrases and in the future may
obviate the need for nucleases.40

Table 1. Biotechnology Differences among Prototype Genome-Editing Techniques

Serial No. Parameter ZFN TALEN CRISPER/Cas Reference

slightly complex (identical repeats are
moderate (ZFNs need customized simpler (available versions for 48
1 design simplicity multiple, which creates technical issues
protein for every DNA sequence) crRNA can be easily designed)
of engineering and delivery into cells)
24,49
2 engineering feasibility low higher highest
high-yield multiplexing available
48,50
3 multiplex genome editing few models few models (no need for obtaining embryonic
stem cells)
progress demonstrated (CRISPR
not much progress (need not much progress (need individual 51
4 large-scale library preparation only requires plasmid containing
individual gene tailoring) gene tailoring)
small oligonucleotides)
24
5 specificity low higher highest
a b 24,48,52
6 efficiency normal normal high
53
7 cost low high low
a
Some new versions are more efficient24,48 but CRISPR science is evolving more.
b
Cpf1 protein addition will probably improve cell delivery methods.51,52

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Table 2. Side Effect Profiles for Genome-Editing Methods Table 3. Clinical and Research Applications across Important Genome-
Editing Techniques
Serial No. Parameter ZFN TALEN CRISPER/Cas Reference
Serial No. Parameter ZFN TALEN CRISPER/Cas Reference
off-target effect
1 – – – 54
60
incidence 1 diagnostic utility + + +++
61
homologous 2 clinical trial use ++ + +++
a recombination + + + –
utility as epigenetic 62
rate frequency 3 ++ +++ ++++
marker
non-homologous
++ (only with 55,56 making gene-knockout 63
b end joining (NHEJ) + + 4 no no yes (CRISPRi)
earlier versions) models for research
mutation rates
capacity for modification 64
immune reaction 57,58 5 no no probable
c less less more of mitochondrial DNA
susceptibility
genetic editing in 65
RNA-guided 6 no no yes
human babies
endonuclease
66
d (RGEN)-induced   ++ 59 7 RNA editing no no yes
off-target
mutatagenesis
2 cytotoxicity chances ++ + + – Conclusions
This review discussed multiples aspects of genome-editing technolo-
gies, including a classification; some basic explanatory concepts on
The most interesting part of the genome-editing technique, which
mechanisms; and comparison between methods, newer advancements,
may be the game changer in genome editing, is the whole genome
and bioethical concerns. It seems that CRISPR/Cas technologies are
engineering by synthesis that in fact would re-create the genome
probably superseding ZFNs and TALENS. However, the CRIPSR/
from scratch as per the given designed DNA code. This probably
Cas methods are also being improvised, and newer additions have
will become the synthetic genomics of the future.41 Though research
further enhanced its functional capabilities with reduced off-target
work in this domain stands preliminary, over time it is anticipated
effects. Furthermore, the process of engineering better gene modifica-
that this technology may overtake the concept of genome editing.
tion technologies is evolving and can one day replace even CRISPR/
Cas, possibly shifting to synthetic genomics. Among all these revolu-
Bioethical Issues and Genome-Editing Techniques
tionary developments, bioethical concerns need serious attention.
Genome-editing tools are powerful in terms of their potential to not
only bring biotechnological revolution in the field of crop develop-
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