CRISPR/Cas-Based Modifications for Therapeutic Applications: A Review

Cas13d: A New Molecular Scissor for Transcriptome Engineering

“…Furthermore, studies have shown some distinct requirement to get optimal Cas13d endoRNase activity such as i) HEPN nuclease activity of Cas13d is strongly depend on the base pairing of target ssRNA i.e., binding of > 21-nucleotide complementarity with crRNA spacer causes full conformational activation and optimal cleavage activity of Cas13d while 18–20 nucleotide complementarity causes half-maximal cleavage activity; ii) presence of any mismatches in the two separate crRNA spacer region (an internal region (spacer nucleotides 5–8) and 3′-end region (spacer nucleotides 13–22) with target ssRNA, founds to be intolerant and completely eradicate the ssRNA cleavage activity; iii) presence of Mg 2+ and additional accessory component WYL domain increases the endoRNase activity; iv) Cas13d tends to cleave only structurally accessible ssRNA sequences, any secondary structure present in the target RNA sequence it fails to recognize and the RNA cleavage activity is abolished; v) Cas13d has exhibited no preference for PFS imposed ssRNA cleavage but has shown considerable preference for uracil bases in target ssRNA structures. Moreover, the vigorous cleavage activity of Cas13d has been found to remain functional through a broad range of temperatures i.e., 24–41°C and thus making it a potential tool for use in transcriptome engineering in a wide range of hosts ( Bharathkumar et al, 2021 ; Feng et al, 2021 ).…”

“…In addition to that nCas9 has been utilized beyond genome editing, as a DNA base editor and also as a gene regulator by fusing nCas9 with base alteration enzyme such as an adenine deaminase, cytidine deaminase, uracil glycosylase inhibitor (UGI), which successfully helps to convert cytidine (C) to thymine (T), adenine (A) to guanine (G), and vice-versa in a targetable manner without altering genome ( Hilton et al, 2015 ; Barrangou and Doudna, 2016 ). Similarly, dCas9 has been utilized in several applications beyond genome editing such as by fusing it with transcriptional activators or repressors to activate or inhibit targeted gene expression, effector proteins to fuse with fluorescent proteins for genome/chromatin imaging, epigenetic modifiers for regulation of epigenetic modification ( Chakravarti et al, 2022 ), and also can be fused with deaminase to function as base-editor to the targeted sites ( Figure 4 ) ( Chen et al, 2014 ; Gilbert et al, 2014 ; Brocken et al, 2018 ; Bharathkumar et al, 2021 ).…”

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

“…Although Cas9 also harbors RuvC and HNH domains, at sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand. In contrast, the Cas9 RuvC-like domain cleaves the noncomplementary strand, thereby stressing the fact that target recognition by Cas9 requires both a seed sequence in the crRNA as well as a GG dinucleotide-containing protospacer adjacent motif (PAM) sequence adjacent to the crRNA-binding region in the DNA target[ 2 , 16 - 20 ]. The majority of type V CRISPR modules can recognize dsDNA targets.…”

“…A similar arrangement in the chromosomes of Gram-negative bacteria ( Escherichia coli, E. coli ) was also reported by Ishino et al in 1987 and was again confirmed in 2010[ 1 ]. The type II CRISPR/Cas system from Streptococcus pyogenes could specifically recognize and cleave target DNA guided by gRNA[ 2 ]; thus, laying the foundation for the development and utilization of the CRISPR/Cas system. Over the past few years, several CRISPR/Cas systems belonging to Cas proteins with different characteristics have been developed, which in turn have produced many CRISPR/Cas system-related toolboxes, offering functional robustness, efficiency, and ease of implementation in multiple organisms[ 3 ].…”

Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2′-fluoro and Locked Nucleic Acid Nucleotides

“…Adaptive immune defense systems of bacteria use clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems to resist foreign invading nucleic acids [ 1 , 2 ]. The CRISPR/Cas9 technology has become a potent instrument for altering the genomes and found extensive applications in molecular biology and genetic engineering [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ].…”