Fig. 3.

Representative processes related to gRNA to improve editing efficiency. (A) crRNA array processing. For multiple targets, the crRNA array can generally be processed by the RNA enzyme activity of Cas (for example, most type V Cas proteins), endogenous tRNA processing enzymes, ribozymes (commonly including HH and HDV ribozymes), and Csy4 RNA enzyme. (B) gRNA engineering by structural modification. Common modifications include extending the 5′ or 3′ terminal, base substitution in the stem-loop structure, formation of chimeric DNA-RNA by replacing part of RNA with DNA, and re-designing artificial gRNA reference to gRNAs derived from Cas orthologs. (C) Chemical modification of crRNA. This mainly involves the modification of the phosphoric acid backbone, namely, PS modification, and sugar modification, mainly including 2′-O-Me, 2′-F, MS, and MSP modifications. Cas: CRISPR-associated proteins; tRNA: transfer RNA; HH: hammerhead; HDV: hepatitis deltavirus; gRNA: guide RNA; PS: phosphorothioate; 2′-O-Me: 2′-O-methyl; 2′-F: 2′-fluoro; MS: 2′-O -methyl -3′phosphorothioate; MSP: 2′-O-methy1-3′thioPACE.