This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 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. 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 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.6,7 However, the technique as of now could not gain widespread introduction due to the emergence 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-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.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 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).8,9 (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, Figure 1. A Consolidated Overview of Genome-Editing Techniques Molecular Therapy: Nucleic Acids Vol. 16 June 2019 327 www.moleculartherapy.org Review which thus results in the formation of 2 segments of DNA fragment.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 engineering 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 off-target effects like reducing site specificity, less DNA integration, and possible host genome mutations.14 (4) Protein-based nuclease systems. These systems incorporate nuclease proteins for DNA sequence editing. The common techniques 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 recognition sites to create nicks, as required for DNA sequence changeExcision of Selective Site of dsDNA by Utilizing Artificial Restriction DNA Cutter 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) rAAVdelivered desirable DNA code replacement of the DNA. 328 Molecular Therapy: Nucleic Acids Vol. 16 June 2019