Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice

Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N₄ GATT and N₄ CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.