Targeted mutagenesis and base editing using engineered Brevibacillus laterosporus Cas9 with expanded target scope in...

The CRISPR/Cas9 system is now widely used for precise genome editing in many crop species, allowing targeted mutagenesis and precise base substitution. While CRISPR/Cas9-mediated genome editing is highly accurate, a limitation of this technology is the requirement for an appropriate protospacer adjacent motif (PAM) for target site recognition. Cas9 from Brevibacillus laterosporus (BlCas9) has been reported to recognize N₄CNDD PAMs with a pronounced preference for A at the 7th and 8th positions of the PAM, and is used for genome editing in maize and human cells. Recently, the enhanced BlCas9 (enBlCas9) variant was reported that includes two amino acid mutations in the PAM-interacting domain of BlCas9 and exhibits enhanced genome editing activity with an expanded target scope in vitro and in human cells. Here, we demonstrate the BlCas9- and enBlCas9-mediated genome editing in rice. Both BlCas9 and enBlCas9 can introduce targeted mutations in rice, and enBlCas9 can broaden the target scope with a non-A residue at the 7th and 8th bases of the PAM. Furthermore, enBlCas9 is applicable for precise base editing with extended target sites, such as C-to-T and A-to-G base conversions. In addition, we developed and validated a proximal CRISPR targeting method (proxy-CRISPR) in which nuclease-dead SpCas9 (SpdCas9) bound near the target sites can improve the genome editing activity of BlCas9 and enBlCas9. These enBlCas9-based genome editing technologies are expected to broaden the scope of efficient targeted mutation and precise base editing in rice.