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 unexp...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 unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More 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.展开更多
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)+1 种基金Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2104)Science and Technology Research Program of Shanghai(19DZ2282100)。
文摘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 unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More 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.
