Discovery and engineering of ChCas12b for precise genome editing

作     者：Jingjing Wei Jingtong Liu Yuwen Tian Ziwen Wang Linghui Hou Yuan Yang Chen Tao Miaomiao Li Bao-Qing Gao Huanyu Zhou Xixi Zheng Junnan Tang Song Gao Li Yang Renjie Chai Yongming Wang 魏京京;刘靓童;田雨闻;王子文;侯凌慧;杨元;陶晨;李苗苗;高宝青;周桓羽;郑茜茜;唐俊楠;高嵩;杨力;柴人杰;王永明

作者机构：Department of CardiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhou 450052China Shanghai Pudong HospitalSchool of Life SciencesShanghai Engineering Research Center of Industrial MicroorganismsFudan UniversityShanghai 200438China State Key Laboratory of Oncology in South ChinaCollaborative Innovation Center for Cancer MedicineSun Yat-sen University Cancer CenterGuangzhou 510060China Shanghai Institute of Nutrition and HealthUniversity of Chinese Academy of SciencesChinese Academy of SciencesShanghai 200031China Center for Molecular MedicineChildren’s Hospital of Fudan University and Shanghai Key Laboratory of Medical EpigeneticsInternational Laboratory of Medical Epigenetics and MetabolismMinistry of Science and TechnologyInstitutes of Biomedical SciencesFudan UniversityShanghai 201102China State Key Laboratory of BioelectronicsDepartment of Otolaryngology Head and Neck SurgeryZhongda HospitalSchool of Life Sciences and TechnologyAdvanced Institute for Life and HealthJiangsu Province High-Tech Key Laboratory for Bio-Medical ResearchSoutheast UniversityNanjing 210096China Co-Innovation Center of NeuroregenerationNantong UniversityNantong 226001China 

出 版 物：《Science Bulletin》 (科学通报（英文版）)

年 卷 期：2024年第69卷第20期

页      面：3260-3271页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：supported by the National Key Research and Development Program of China(2021YFC2701103,2021YFA0910602,and 2019YFA0802804) the National Natural Science Foundation of China(82070258 and 31925011) Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2104) Science and Technology Research Program of Shanghai(19DZ2282100) 

主　　题：Genomeediting CRISPR/Cas12b ChCas12b-D496A Allele-specific disruption 

摘      要：Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely *** this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active *** hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting *** optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous ***,we identified nine mutations enhancing ChCas12b *** importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.