Genome engineering using the CRISPR/Cas system

Figure 1 Schematic of the principles of clustered regularly at interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas)-mediated genome editing. The CRISPR associated 9 (Cas9) endonuclease can generate sequence-specific double strand breaks (DSBs) of target DNAs bound to small guide RNAs (sgRNAs). The binding site of a target DNA requires a protospacer-adjacent motif (PAM) (with the sequence NGG). DSBs generated by the Cas9 endonuclease are repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Figure 2 Improvement of site-specificity by double nicking. A: Double strand breaks (DSBs) using wild-type interspaced short palindromic repeats associated 9 (Cas9) endonuclease; B: DSBs using a pair of small guide RNAs (sgRNAs) guiding Cas9 D10A nickases. Using paired nicking can reduce off-target activity because individual nicks that unexpectedly occurred at off-target sites are predominantly repaired by the high-fidelity base excision repair pathway.