Highly efficient CRISPR-SaKKH tools for plant multiplex cytosine base editing

Base editing, as an expanded clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus (SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif (PAM) that the canonical Streptococcus pyogenes Cas9 (SpCas9) or its variants (e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor (SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.