Engineered Cas9 variant tools expand targeting scope of genome and base editing in rice.

Recently, some newly engineered SpCas9 variants, such as xCas9 3.7 and Cas9-NG, expand the PAM recognition site to NG and show function efficiency in mammalian cells. However, there is still not enough comparative data for their efficiencies on the genome and base editing in plants. In addition, there is also a lack of enhanced specific Cas9 variant with NG PAM. Here, we developed a serial of genome editing tools using different Cas9 variants containing the xCas9, the Cas9-NG and the enhanced specific Cas9-NG (eCas9-NG), and of cytosine base editors (CBE 4 editors, xCas9n-CBE, Cas9n-NG-CBE and eCas9n-NG-CBE) and adenine base editors (ABE 7.10 editors, xCas9n-ABE, Cas9n-NG-ABE and eCas9n-NG-ABE). We systematically investigated the editing activities of these tools with expending target scope in transgenic rice. Our results indicated that the Cas9-NG and eCas9-NG variants enable more efficient genome editing and targeted C-T single nucleotide substitutions with extended non-canonical PAMs than those of xCas9. But their ABE editors worked inefficiently in rice. The Cas9-NG variant is more suitable for genome engineering in rice. Taken together, our work has engineered and yielded Cas9 variant tools with a broadened PAM compatibility, which will facilitate plant functional genome and crop genetic improvement.