# Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice

## TL;DR

Researchers improved genome editing in rice by creating a new chimeric Cas9 variant with better efficiency and broader target range.

## Contribution

A novel chimeric SpcRN++ Cas9 variant is engineered with enhanced editing efficiency and compatibility with base editors in plants.

## Key findings

- SpcRN++ shows higher genome editing efficiency than Sc++ in rice protoplasts and transgenic plants.
- SpcRN++-based base editors improve cytosine and adenine editing in plants.
- Herbicide-resistant rice was successfully generated using SpcRN++.

## Abstract

The Streptococcus canis Cas9 protein (ScCas9) recognizes the NNG protospacer adjacent motif (PAM), offering a wider range of targets than that offered by the commonly used S. pyogenes Cas9 protein (SpCas9). However, both ScCas9 and its evolved Sc++ variant still exhibit low genome editing efficiency in plants, particularly at the less preferred NTG and NCG PAM targets. In this study, a chimeric SpcRN++ variant is engineered by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the R221K/N394K mutations, and retaining the positively charged loop of S. anginosus Cas9. The SpcRN++ variant exhibits a higher genome editing capacity and wider target range than the Sc++ variant in rice protoplasts and stable transgenic plants. Further evidence indicates that nSpcRN++‐based A3A/Y130F and TadA8e exhibit enhanced cytosine and adenine editing efficiency in plants. Finally, herbicide‐resistant rice germplasms are produced by targeting the OsACC gene using nSpcRN++‐based adenine base editors. These results demonstrate that SpcRN++ is a powerful tool for genome editing in plants, and this integrative protein engineering strategy holds promise for engineering other Cas9 proteins.

This study engineers a chimeric SpcRN++ variant by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the evolutionary R221K/N394K mutations, and retaining the positively charged loop from S. anginosus Cas9. SpcRN++ demonstrates superior genome editing capability than Sc++ in rice. SpcRN++ also exhibits high compatibility with base editing tools, making it suitable for trait improvement applications.