Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM. Then, engineered and natural Cas variants with altered PAM recognition are used for base editor to expand the flexibility of base substitution position. In this chapter, we describe a protocol for base editing based on SpCas9-NG, which is a rationally engineered SpCas9 variant that can recognize relaxed NG PAMs.