www.moleculartherapy.org Review 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 combinatorial approach where restriction endonucleases are joined with zinc-finger-binding domain protein. Figure 4 explains their mechanism of action in detail, where a binding protein domain identifies 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 codons 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 system, adopted in nature by some prokaryotic cells like Archea and probably some bacteria.21 CRISPR in itself has two components, 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 bacterium and can be re-activated once encountered with a similar foreign antigen. CRI has similar nucleotides (repeats) representing 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 double-stranded DNA (dsDNA) nicks at the guided selected sites, causing a site-specific cleavage and, thus, destruction of the antigen.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 conditions.25–27 At the time of writing this review, the news broke about 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. Molecular Therapy: Nucleic Acids Vol. 16 June 2019 329 www.moleculartherapy.org Review 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 engineered, 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 complementary 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 nucleotides.29 Over the last few years, CRISPR/Cas9 technology has gained widespread popularity on account of its simplicity and specificity, with different versions of the original now under research. Multiple experimentations 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 under the guidance of crRNA to target DNA, RNase III, trans activating 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,