A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing.

Yao Wang, Tao Qi, Jingtong Liu, Yuan Yang, Ziwen Wang, Ying Wang, Tianyi Wang, Miaomiao Li, Mingqing Li, Daru Lu, Alex Chia Yu Chang, Li Yang, Song Gao, Yongming Wang, Feng Lan

Journal: Science advances 2023;9(6):eabo6405

PMID: 36763662

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Abstract

The CRISPR-Cas system can treat autosomal dominant diseases by nonhomologous end joining (NHEJ) gene disruption of mutant alleles. However, many single-nucleotide mutations cannot be discriminated from wild-type alleles by current CRISPR-Cas systems. Here, we functionally screened six Cas12j nucleases and determined Cas12j-8 as an ideal genome editor with a hypercompact size. Cas12j-8 displayed comparable activity to AsCas12a and Un1Cas12f1. Cas12j-8 is a highly specific nuclease sensitive to single-nucleotide mismatches in the protospacer adjacent motif (PAM)-proximal region. We experimentally proved that Cas12j-8 enabled allele-specific disruption of genes with a single-nucleotide polymorphism (SNP). Cas12j-8 recognizes a simple TTN PAM that provides for high target site density. In silico analysis reveals that Cas12j-8 enables allele-specific disruption of 25,931 clinically relevant variants in the ClinVar database, and 485,130,147 SNPs in the dbSNP database. Therefore, Cas12j-8 would be particularly suitable for therapeutic applications.

Address: State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.; State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China.; CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.; Laboratory for Reproductive Immunology, NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Hospital of Obstetrics and Gynecology, Shanghai Medical School, Fudan University, Shanghai 200080, China.; NHC Key Laboratory of Birth Defects and Reproductive Health, Chongqing Key Laboratory of Birth Defects and Reproductive Health, Chongqing Population and Family Planning, Science and Technology Research Institute, Chongqing 400020, China.; Department of Cardiology and Shanghai Institute Precision Medicine, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200125, China.; Center for Molecular Medicine, Children's Hospital, Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 201102, China.; Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, China.; State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.

DOI: 10.1126/sciadv.abo6405