Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Gene Editing Technologies

Background:

• The Streptococcus canis Cas9 protein (ScCas9) offers a broader protospacer adjacent motif (PAM) recognition (NNG) than Streptococcus pyogenes Cas9 (SpCas9).
• Existing ScCas9 and its variant Sc++ show limited genome editing efficiency in plants, especially at NTG and NCG PAM sites.

Purpose of the Study:

• To engineer a novel chimeric Cas9 variant with improved genome editing efficiency and a wider target range in plants.
• To evaluate the efficacy of the engineered variant and its derivatives for base editing and generating crop traits.

Main Methods:

• Engineered a chimeric SpcRN++ variant by combining the SpCas9 recognition (REC) domain with the Sc++ variant, incorporating specific mutations (R221K/N394K) and the S. anginosus Cas9 positively charged loop.
• Assessed genome editing capacity and target range in rice protoplasts and stable transgenic plants.
• Tested nSpcRN++-based adenine base editors (A3A/Y130F and TadA8e) for cytosine and adenine editing.
• Generated herbicide-resistant rice by targeting the OsACC gene using nSpcRN++-based adenine base editors.

Main Results:

• The SpcRN++ variant demonstrated significantly higher genome editing capacity and a broader target range compared to the Sc++ variant in rice.
• nSpcRN++-based base editors (A3A/Y130F and TadA8e) showed enhanced cytosine and adenine editing efficiency in plants.
• Successfully produced herbicide-resistant rice germplasms using nSpcRN++-based adenine base editors targeting the OsACC gene.

Conclusions:

• The engineered SpcRN++ variant is a potent tool for advancing plant genome editing.
• This protein engineering approach provides a versatile strategy for developing improved Cas9 variants for diverse plant applications.
• The development of herbicide-resistant rice highlights the practical utility of SpcRN++ in crop improvement.