High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

Autor:	Yuen CTL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Thean DGL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Chan BKC; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Zhou P; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Kwok CCS; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Chu HY; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Cheung MSH; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Wang B; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Chan YM; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong SAR, China., Mak SYL; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong SAR, China., Leung AY; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China.; Division of Haematology, Department of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; The Jockey Club Centre for Clinical Innovation and Discovery, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China., Choi GCG; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Zheng Z; Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong SAR, China.; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China.; Biotechnology and Health Centre, City University of Hong Kong Shenzhen Research Institute, Shenzhen, China., Wong ASL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China.; Department of Electrical and Electronic Engineering, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2022 Feb 22; Vol. 50 (3), pp. 1650-1660.
DOI:	10.1093/nar/gkab1291
Abstrakt:	The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity. (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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