Rationally designed miCas9 mediates gene editing

In this study, we report the development of meticulous-integration Cas9 (miCas9) by fusing a minimal motif consisting of thirty-six amino acids encoded by BRCA2 Exon 27 (Brex27) to spCas9. In comparison to spCas9, miCas9 enhances double-stranded DNA (dsDNA) mediated large size gene knock-in (ds-KI) rates, systematically reduces off-target indel events, maintains or increases single-stranded oligodeoxynucleotides (ssODN) mediated precise gene editing (ss-PGE) rates, and effectively reduces on-target indel rates in both ds-KI and ss-PGE applications. Furthermore, we demonstrate that Brex27 can work as a plug and play module, compatible and synergistic with other Cas9 variants.