FIG 2.

Application of CRISPR/Cas9 in targeted genome editing. (A) Widely used S. pyogenes Cas9 with the HNH and RuvC domains is directed by tracrRNA-crRNA duplexes or gRNA to cut the complementary or noncomplementary strand, respectively. Cuts are made at the positions (indicated by red arrows) that are 3 bp upstream of PAM sites (purple characters). All components required for RNA-guided genome editing in foreign hosts are expressed by delivering coexpression plasmids, DNA expression cassette fragments, or sole RNA transcripts (B). Expressed tracrRNA-crRNA duplexes or gRNA is assembled with Cas9, generating executive complexes. These complexes generate breaks in the genome that may lead to cell death if the DSBs are not removed (①), induce error-prone nonhomologous end joining (NHEJ) to rejoin the ends and introduce undefined small deletions and additions (indels) (②), or trigger homology-directed repair (HDR) when homology-containing dsDNA or ssDNA templates are given (③) to confer precise DNA substitution, deletion, or insertion.