Abstract
CRISPR genome editing technologies have been improving by every passing day. The initial CRISPR/Cas9 technologies, though emerged an improved version of genome editing in competition with TALENS and ZFNs, was nevertheless not free from technical and off-target effects. Technological improvements overtime start addressing issues with original CRISPR/Cas9 technology. The major areas of improvement targeted nucleases and delivery methods. Overtime the nuclease like Cas9 had some modifications like FokI-dCas9, Truncated guide RNAs (tru-gRNAs), Paired Cas9 nickase, Cpf1, Cas6 with Csm/Csr complex and chemically treated Cas9. In terms of delivery methods the improvements came along after almost all methods including viral methods like Recombinant Adeno Associated Viruses (rAAV), Lentivirus (LV), and bacteriophages. The review summarizes various non-viral gene delivery modes including physical methods like electroporation and chemical methods like nano particles, cell-derived membrane vesicles (CMVs) with upgraded developments. The review also compares various modes of delivering CRISPR gene editing machinery.
Highlights
Cluster Regularly Interspaced Short Palindromic Repeat (CRISPR) currently emerged as the standalone technology in the field of genome editing
Later various pieces were joined through biotechnology to shape the system i.e., CRISPR/Cas9 to edit genome by the help of a simplified guide RNA formed by fusion of tracrRNA (Trans activating CRISPR RNA) and crRNA which acting as a duplex with ability to guide Cas9 accurately to its cleavage site on DNA strand
Provided the previously in vogue Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs) showed much promise as gene editing options, still CRISPR technology was able provide better alternative as it did not require reengineering the enzyme for every new target sequence. [4, 5] the limitations, lesser efficiency and reengineering issues with earlier genome editing techniques led to the rise of CRISPR/Cas9 system by virtue of its feasibility of engineering, cost-effectiveness and measurability
Summary
Cluster Regularly Interspaced Short Palindromic Repeat (CRISPR) currently emerged as the standalone technology in the field of genome editing. [3] CRISPR Associated protein-9 with inherent endonuclease function assisted by “gRNA” was able to efficiently create specific double-helical breaks in the DNA, further improving gene editing quality [3]. Provided the previously in vogue Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs) showed much promise as gene editing options, still CRISPR technology was able provide better alternative as it did not require reengineering the enzyme for every new target sequence. The initial technology as devised by Jennifer Doudona and Emmanuelle Charpentier suffered due to multiple off-target effects including: a-chromosomal translocations and random mutations in general [7], b-immunogenicity related to Cas proteins, sgRNA and sometimes with inserted DNA fragments[8], c-efficiency in CRISPR/Cas payload delivery into cell [9], appearance of resistance after successful CRISPR/Cas use as therapeutic strategy to eradicate certain infections like HIV [10], and the inability of conventional CRISPR/Cas technology to edit PAM free sequence and sometimes CRISPR-resistant systems [11].
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