Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing Techniques and Bioethical Concerns for Clinical Application

The traditional healthcare system is at the doorstep for entering into the arena of molecular medicine. The enormous knowledge 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-editing 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 interfering RNA (siRNA), have been widely adopted in the research laboratories to replace lab animals and cell lines. The latest discovery 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 engineering. This review generally discusses the various gene-editing technologies in terms of the mechanisms of action, advantages, and side effects. The traditional healthcare system is at the doorstep for entering into the arena of molecular medicine. The enormous knowledge 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-editing 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 interfering RNA (siRNA), have been widely adopted in the research laboratories to replace lab animals and cell lines. The latest discovery 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 engineering. 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 molecular technologies, which are now heading forward toward translation into clinical and laboratory practice.1Friedrich M. Ueber die chemische Zusammensetzung der Eiterzellen [On the chemical composition of pus cells].Medicinisch-chemische Untersuchungen. 1871; 4: 441-460Google Scholar 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 scientists to manipulate the genetic codes at various levels.2Watson J.D. Crick F.H.C. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (8367) Google Scholar Over the last two decades we saw a plethora of molecular techniques, which allowed 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.3Sanger F. Coulson A.R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase.J. Mol. Biol. 1975; 94: 441-448Crossref PubMed Scopus (1587) Google Scholar 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.4Mullis K.B. https://www.karymullis.com/Date: 2016Google Scholar, 5Pavletich N.P. Pabo C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref PubMed Scopus (1744) Google Scholar Common to these methods are three basic steps, including mechanisms for genetic tool entry into the cell and later nucleus; altering gene transcription and onward processing function; and, finally, the end-output in the shape of a suppressed, overexpressed, or simply an altered protein product.6Claussin C. Chang M. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae.PLoS Genet. 2016; 12: e1006176Crossref PubMed Scopus (16) Google Scholar, 7Arai N. Kagawa W. [Molecular mechanisms of homologous recombination promoted by budding yeast Rad52].Seikagaku. 2014; 86: 693-697PubMed Google Scholar From a holistic point of view, the techniques involve an apparently simplistic concept involving multiple receptor-ligand interactions; varying cell entry modes like lipofection, sonification, and transfection; and further downstream pathway effects. 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 platforms for these technologies are improving every day, with a plethora of new data appearing due to technology miniaturization and automation 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 imprecision, defining the indications and contraindications of every technique to help improve the concept of personalized medicine. This review briefly explains the available technologies, provides comparison and contrast between different genome-editing methods, and identifies some newer versions of genome editing with possible bioethical concerns. 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 references. Specific searches for articles dealing with specific genome-editing methods included conventional genome-editing systems (n = 100), chemical methods (n = 252), meganucleases (n = 83), zinc-finger nucleases (ZFNs) (n = 890), transcription activator-like effector nucleases (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 comparative 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. 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 techniques. 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 therefore has been considered as a therapy target in certain cancers like BRCA 1 and 2 repair pathways.6Claussin C. Chang M. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae.PLoS Genet. 2016; 12: e1006176Crossref PubMed Scopus (16) Google Scholar, 7Arai N. Kagawa W. [Molecular mechanisms of homologous recombination promoted by budding yeast Rad52].Seikagaku. 2014; 86: 693-697PubMed Google Scholar However, the technique as of now could not gain widespread introduction due to the emergence of newer techniques.(2)Chemical modalities of genome editing. Komiyama8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar utilized non-restriction enzyme methodology termed artificial restriction DNA cutter (ARCUT). This method uses pseudo-complementary 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.8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar 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 introduction, the technique looked quite appealing to the clinical market; however, later issues like increased turnaround time and specifically the manufacturing of site-specific pcPNA became huge hurdles (Figure 2).8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar, 9Ito K. Komiyama M. Site-selective scission of human genome using PNA-based artificial restriction DNA cutter.Methods Mol. Biol. 2014; 1050: 111-120Crossref PubMed Scopus (7) Google Scholar(3)Homing endonuclease systems. Homing endocucleases (HEs) with this word “homing” practically is interpreted as lateral transmission of a genome DNA sequence. The general concept involves a DNA segment where a site is removed by the endocnulceases, which thus results in the formation of 2 segments of DNA fragment.10Schultz B.R. Chamberlain J.S. Recombinant adeno-associated virus transduction and integration.Mol. Ther. 2008; 16: 1189-1199Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar 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.11Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. et al.A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (246) Google Scholar 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 3.12Yoon Y. Wang D. Tai P.W.L. Riley J. Gao G. Rivera-Pérez J.A. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.Nat. Commun. 2018; 9: 412Crossref PubMed Scopus (41) Google Scholar Issues pertaining to this technology include engineering difficulties in the preparation of these nucleases as well as developing vectors for their entry into cells.13Rey-Rico A. Cucchiarini M. Controlled release strategies for rAAV-mediated gene delivery.Acta Biomater. 2016; 29: 1-10Crossref PubMed Scopus (37) Google Scholar Another issue with rAAV, though improving with better biotechnology, was off-target effects like reducing site specificity, less DNA integration, and possible host genome mutations.14Jin L. Lange W. Kempmann A. Maybeck V. Günther A. Gruteser N. Baumann A. Offenhäusser A. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.J. Biotechnol. 2016; 233: 171-180Crossref PubMed Scopus (9) Google ScholarFigure 3Schematic Showing within with and with DNA for include the entry of into cell, by and within release of for entry into delivery of homing and desirable DNA of the DNA and (6) desirable DNA of the Figure nuclease systems. These systems nuclease for DNA editing. The common techniques are termed molecular DNA these are that are in the to of DNA sequences was as a genetic tool to modify This genetic has been in by the to create as for DNA These meganucleases are by to create like and which can further site-specific M. C. P. Duclert A. of meganucleases using a learning 2014; PubMed Scopus (9) Google Scholar The technique two basic first is the of a cleavage site, and endonucleases out the K. G. site cleavage of homing Acids Res. 2018; Google Scholar The related to meganucleases is less as they are and specific site However, are newer techniques now in the clinical arena that have not allowed them to are artificial by a combinatorial approach where restriction endonucleases are with Figure explains their of in where a protein identifies after the desirable site, which is at a specific by restriction endonucleases The biotechnology is in terms of with on side of the DNA The technique in recent years has widespread due to its simplicity and specificity, and it is in clinical for certain and of Zinc Mol. Biol. PubMed Scopus Google Scholar, P. Wang Y. Y. et by from the in and Ther. 2018; 12: Full Text Full Text PDF PubMed Scopus Google ScholarFigure Showing by mechanisms include the endonucleases and are into the cell, and are to the attach with DNA to be out the DNA segment by double-stranded DNA and the desirable DNA segment is and into the DNA Figure almost in terms of manufacturing and of They are by a where a restriction nuclease is to a protein G. J. Mol. Biol. PubMed Scopus (9) Google Scholar The between and is that the can target in and the can 1 thus slightly more site specific with off-target S. D. of for Mol. Biol. 2016; PubMed Scopus Google Scholar However, the techniques (Figure Showing Mechanisms of of gene editing include the endonucleases and are into the cell, and are to the the DNA segments and with the DNA and after the DNA segments are the desirable segment of DNA is into the Figure DNA systems. These systems include the different of CRISPR methods. The concept of CRISPR is and has been from an adopted in by some cells like and some S. J. F. M. A. immune system can multiple to target Biol. Full Text Full Text PDF PubMed Scopus Google Scholar CRISPR in has two including termed as which are by varying and and of them a to foreign The may the genetic for a and can be with a foreign has like between different A. the of CRISPR/Cas9 from to Mol. 2016; PubMed Scopus Google Scholar Figure attempts to a basic overview of the CRISPR/Cas9 the of CRISPR/Cas9 to the of at the by the Figure as depicted in Figure has a nuclease CRISPR RNA also termed RNA the protein a possible like a it with the help of double-stranded DNA at the a site-specific cleavage and, thus, of the F. Y. CRISPR/Cas9 for genome and Mol. Genet. 2014; PubMed Scopus Google Scholar Moreover, the from the is as within P. M. A. of genome editing through 2016; PubMed Scopus Google physiological of as been utilized for multiple clinical W. J. N. S. A genome editing to human genetic PubMed Scopus Google Scholar, L. W. S. of CRISPR/Cas9 by strategies for gene Google Scholar, S. Y. J. of CRISPR/Cas9 technologies with in the and of Genet. 2018; PubMed Scopus Google Scholar the time of this the about and to be the first where the human genome was edited to create A. scientists are CRISPR A is to create the first whose DNA has been using gene editing. Scholar Specific has been which can be into cell and later where the is associated with the after provided by the specific This between and the segment to the DNA In clinical and research the can be specifically by the of to change the of M. A at genome genome Ther. Full Text Full Text PDF PubMed Scopus (16) Google the last CRISPR/Cas9 technology has widespread on of its simplicity and specificity, with different versions of the now Multiple and biotechnologies have been the technologies into of based on processing and further action, including the 1 system. This utilized or for of further cleavage needs and for 2 system. the of to target RNA and a protein are in at the system. the 1 this uses for processing The of this technique is its of which is by a specific complex D. L. A. M. The immune mechanisms and PubMed Scopus Google from the conventional of the data review provided multiple biotechnologies now include CRISPR A system for of gene Biol. PubMed Scopus Google Scholar F. A for genome editing and transcription Biotechnol. PubMed Scopus Google Scholar and modifications like P. J. M. S. L. for target specificity and for genome Biotechnol. PubMed Scopus Google techniques. These methods may not genome editing, but they are capable of the DNA sequence. These technologies include CRISPR and RNA and Mol. Biol. PubMed Scopus Google Scholar, M. N. Y. of an for of cell 2018; 9: PubMed Scopus Google Scholar, S. of and 2016; PubMed Scopus Google Scholar The include the entry of into cell, by and within release of for entry into delivery of homing and desirable DNA of the DNA and (6) desirable DNA of the The mechanisms include the endonucleases and are into the cell, and are to the attach with DNA to be out the DNA segment by double-stranded DNA and the desirable DNA segment is and into the DNA sequence. The of gene editing include the endonucleases and are into the cell, and are to the the DNA segments and with the DNA and after the DNA segments are the desirable segment of DNA is into the The provides a of the various used genome-editing techniques. is enormous and of newer to edit the we further on the comparison of common techniques, including and CRISPR methods. 1, and a comparative these need protein for every DNA complex are which issues of engineering and delivery into versions for can be M. CRISPR/Cas9 in and 2016; PubMed Scopus Google P. M. A. of genome editing through 2016; PubMed Scopus Google Scholar, J. G. genome editing in the CRISPR Biol. PubMed Scopus Google genome available need for M. CRISPR/Cas9 in and 2016; PubMed Scopus Google Scholar, A. S. K. genome engineering in human cells using CRISPR/Cas9 2014; 4: PubMed Scopus Google much gene much gene small CRISPR and Mol. Biol. PubMed Scopus Google P. M. A. of genome editing through 2016; PubMed Scopus Google new versions are more but CRISPR is evolving protein improve cell delivery P. M. A. of genome editing through 2016; PubMed Scopus Google Scholar, M. CRISPR/Cas9 in and 2016; PubMed Scopus Google Scholar, J. S. D. Y. A of J. 2018; PubMed Scopus Google L. L. and with Biol. 2018; PubMed Scopus Google new versions are more P. M. A. of genome editing through 2016; PubMed Scopus Google Scholar, M. CRISPR/Cas9 in and 2016; PubMed Scopus Google Scholar but CRISPR is evolving protein improve cell delivery CRISPR and Mol. Biol. PubMed Scopus Google Scholar, J. S. D. Y. A of J. 2018; PubMed Scopus Google Scholar in a new for J. J.S. and of PubMed Scopus Google recombination with earlier J. and repair pathway Genet. PubMed Scopus Google Scholar, W. S. M. V. editing with in a and Biotechnol. 2014; PubMed Scopus Google CRISPR use of CRISPR for gene 2016; PubMed Scopus Google Scholar, to CRISPR and Biol. 2018; PubMed Scopus Google endonuclease off-target S. Y. J. S. J.S. of off-target effects of endonucleases and Res. 2014; PubMed Scopus Google in a new and W. Wang F. G. Wang elements for and a genetic Genet. PubMed Scopus Google M. A. A. S. M. and strategies for functional in cell PubMed Scopus Google as A. P. V. L. P. M. V. the system for DNA Acids Res. 2016; PubMed Scopus Google for J. L. Y. L. D. of for of in 2016; PubMed Scopus Google for of S. C. C. A. CRISPR/Cas9 and gene techniques and ethical J. 2016; 5: Google editing in human research in human embryos 2016; PubMed Scopus Google C. of The 2016Google Scholar in a new The biotechnology is with a of newer modalities to edit the can be utilized with double-stranded transcription to as a target for multiple diseases, which can the transcription and thus in the change in transcription and further downstream protein M. A PubMed Scopus Google Scholar et J.S. gene-editing mechanisms and Biol. Ther. 12: PubMed Scopus Google Scholar have utilized to for small point a genome-editing This concept involves a which is carried into the cell by a specific providing us with specific and efficient editing of the This technique not need the conventional and, thus, it is termed genome J.S. gene-editing mechanisms and Biol. Ther. 12: PubMed Scopus Google Scholar The technique seems to have off-target effects and is considered to be a modalities of genome editing are also appearing in the with modifications of techniques. et G. and the The for the Ther. 2016; PubMed Scopus Google Scholar used DNA and in gene editing and gene editing. have utilized certain like and in the may the need for A to the to edit a 2016; PubMed Scopus (7) Google Scholar The of the genome-editing which may be the in genome editing, is the genome engineering by synthesis that in the genome from as the DNA This the of the S. S. Y. J. genome engineering by 2018; PubMed Scopus (16) Google Scholar research work in this time it is that this technology may the concept of genome editing. tools are in terms of their to not in the field of and human but in the lead to and in multiple including manipulation of bioethical concerns have been by K. and 2016; PubMed Scopus Google Scholar While time be the of these technologies as or the methods can the human in the and human may be in we not K. N. S. 2018; Google Scholar concerns from include the the the to ongoing clinical about the possible of and F. Issues CRISPR Issues Mol. Biol. 2018; PubMed Scopus (9) Google Scholar, A human PubMed Scopus Google Scholar, The gene-editing of 2018; PubMed Scopus Google Scholar Moreover, the literature also in the of genome editing as a of and the need for by the Genet. 29: PubMed Scopus Google Scholar While the for a and of possible have been in the seems and The therefore a and translation of of technologies for molecular and and This need in of of bioethical regulatory within and and for the allowed limited application. This review discussed of genome-editing technologies, including a some basic on and comparison between methods, newer and bioethical concerns. seems that technologies are and However, the methods are also and newer have further its functional with off-target effects. Furthermore, the of engineering better gene technologies is evolving and can replace to these bioethical concerns need