Gene-editing should be abolished (Due 40 hours)

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.

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: [email protected]

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-

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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),

uthor(s). ://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. A Consolidated Overview of Genome-Editing Techniques

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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 bioethicalconcerns 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

Figure 2. Excision of Selective Site of dsDNA by Utilizing Artificial Restriction DNA Cutter

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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.

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.

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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.

330

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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.

Molecular Therapy: Nucleic Acids Vol. 16 June 2019

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-silencingtechniques. These methodsmaynotfalltrulyunder 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

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

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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

Table 1. Biotechnology Differences among Prototype Genome-Editing Techniq

Serial No. Parameter ZFN TALEN

1 design simplicity moderate (ZFNs need customized protein for every DNA sequence)

slightly multip of engi

2 engineering feasibility low higher

3 multiplex genome editing few models few mo

4 large-scale library preparation not much progress (need individual gene tailoring)

not mu gene ta

5 specificity low higher

6 efficiency normala norma

7 cost low high

aSome new versions are more efficient24,48 but CRISPR science is evolving more. bCpf1 protein addition will probably improve cell delivery methods.51,52

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

ues

CRISPER/Cas Reference

complex (identical repeats are le, which creates technical issues neering and delivery into cells)

simpler (available versions for crRNA can be easily designed)

48

highest 24,49

dels high-yield multiplexing available (no need for obtaining embryonic stem cells)

48,50

ch progress (need individual iloring)

progress demonstrated (CRISPR only requires plasmid containing small oligonucleotides)

51

highest 24

lb high 24,48,52

low 53

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Table 2. Side Effect Profiles for Genome-Editing Methods

Serial No. Parameter ZFN TALEN CRISPER/Cas Reference

1 off-target effect incidence

– – – 54

a homologous recombination rate frequency

+ + + –

b non-homologous end joining (NHEJ) mutation rates

+ + ++ (only with earlier versions)

55,56

c immune reaction susceptibility

less less more 57,58

d

RNA-guided endonuclease (RGEN)-induced off-target mutatagenesis

� � ++ 59

2 cytotoxicity chances ++ + + –

Table 3. Clinical and Research Applications across Important Genome-

Editing Techniques

Serial No. Parameter ZFN TALEN CRISPER/Cas Reference

1 diagnostic utility + + +++ 60

2 clinical trial use ++ + +++ 61

3 utility as epigenetic marker

++ +++ ++++ 62

4 making gene-knockout models for research

no no yes (CRISPRi) 63

5 capacity for modification of mitochondrial DNA

no no probable 64

6 genetic editing in human babies

no no yes 65

7 RNA editing no no yes 66

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The most interesting part of the genome-editing technique, which may be the game changer in genome editing, is the whole genome engineering by synthesis that in fact would re-create the genome from scratch as per the given designed DNA code. This probably will become the synthetic genomics of the future.41 Though research work in this domain stands preliminary, over time it is anticipated that this technology may overtake the concept of genome editing.

Bioethical Issues and Genome-Editing Techniques

Genome-editing tools are powerful in terms of their potential to not only bring biotechnological revolution in the field of crop develop- ment and human pathology but also, in the wrong hands, lead to abuse and misuse in multiple ways, including manipulation of germ- line genetics. Genuine bioethical concerns have been raised by many experts.42 While time will be the actual judge of these technologies as boon or bane, still the methods can impact the human race probably in the most nuclear ways, and our incoming human race may be victimized in ways we do not yet understand.43 Principal concerns apart from illegal germline mutation include the morality, the eu- genics helping the fittest to survive, ongoing clinical debates about informed consent, religious debate, the possible rise of clones, designer babies, and possibly superhumans.44–46 Moreover, the cur- rent literature also rules in the possibility of genome editing as a future weapon of war.47

While the quest for a healthy baby and right of best possible treatment choice have been acknowledged in many societies, the approaching biotechnological revolution seems imminent and undeniable. The pressing need, therefore demands a harmonious and regulated translation of needed aspects of genome-editing-related technologies for molecular medicine and other non-clinical crop and food indus- tries. This will need consensus in public opinion, debates among experts, involvement of biotechnologists, opinions of bioethical ex- perts, regulatory frameworks within legislatures, and final guidelines and oversight for the finally allowed limited application.

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Conclusions

This review discussed multiples aspects of genome-editing technolo- gies, including a classification; some basic explanatory concepts on mechanisms;andcomparisonbetween methods,newer advancements, and bioethical concerns. It seems that CRISPR/Cas technologies are probably superseding ZFNs and TALENS. However, the CRIPSR/ Cas methods are also being improvised, and newer additions have further enhanced its functional capabilities with reduced off-target effects. Furthermore, the process of engineering better gene modifica- tion technologies is evolving and can one day replace even CRISPR/ Cas, possibly shifting to synthetic genomics. Among all these revolu- tionary developments, bioethical concerns need serious attention.

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