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 notfall 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-editing techniques. Though there is enormous development, innovation, and design of newer ways to edit the genome, we focus our further discussion on the comparison of common techniques, including ZFN, TALEN, and CRISPR methods. Tables 1, 2, and 3 provide a comparative 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 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 Molecular Therapy: Nucleic Acids Vol. 16 June 2019 www.moleculartherapy.org Review double-stranded transcription factor decoy (TFD) to act as a therapeutic 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 utilized 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. MartínezGálvez 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 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 Table 1. Biotechnology Differences among Prototype Genome-Editing Techniques Serial No. Parameter ZFN TALEN CRISPER/Cas Reference 1 design simplicity moderate (ZFNs need customized protein for every DNA sequence) slightly complex (identical repeats are multiple, which creates technical issues of engineering and delivery into cells) simpler (available versions for crRNA can be easily designed) 48 2 engineering feasibility low higher highest 24,49 3 multiplex genome editing few models few models high-yield multiplexing available (no need for obtaining embryonic stem cells) 48,50 4 large-scale library preparation not much progress (need individual gene tailoring) not much progress (need individual gene tailoring) progress demonstrated (CRISPR only requires plasmid containing small oligonucleotides) 51 5 specificity low higher highest 24 6 efficiency normala normalb high 24,48,52 7 cost low high low 53 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 Molecular Therapy: Nucleic Acids Vol. 16 June 2019 331 www.moleculartherapy.org Review 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 development and human pathology but also, in the wrong hands, lead to abuse and misuse in multiple ways, including manipulation of germline 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