Background:Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing.The Multiple copy numbers of the MS2 binding site(MBS)can recruit multiple MS2 coat proteins(MCPs),which are usually applied t...Background:Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing.The Multiple copy numbers of the MS2 binding site(MBS)can recruit multiple MS2 coat proteins(MCPs),which are usually applied to amplify signals.Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site.Methods:Multiple copy numbers of the MS2 were ligated to the 3′‐end of sgRNA,and MCP was fused to the 5′‐end of cytosine deaminases.The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions(C‐T)at the target site.Different Cas9 variants,different cytosine deaminases and different copy numbers of MS2 were tested in this system,and the different versions of base editors were compared by editing efficiency and window.Results:In this study,dCas9,nCas9(D10A)and nCas9(H840A)were used.Among these 3 Cas9 variants,dCas9 exhibited higher base mutation efficiency.Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID.We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12.Disappointingly,the sgRNA‐12x MS2 did not improve the editing efficiency or increase the editing window.Conclusion:An optimal version of base editor based on the MS2 system,MS2‐BErAPOBEC1(sgRNA‐2x MS2,MCP‐rAPOBEC1 and dCas9),was obtained.This tool can simultaneously mutate multiple bases at the target site,providing a new approach for the study of genome functions.展开更多
Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a lon...Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.展开更多
基金National Key R&D Program,Grant/Award Number:2017YFC1001903Major Technological Innovation Plan of the Hospital,Grant/Award Number:SWH2016ZDCX1010
文摘Background:Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing.The Multiple copy numbers of the MS2 binding site(MBS)can recruit multiple MS2 coat proteins(MCPs),which are usually applied to amplify signals.Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site.Methods:Multiple copy numbers of the MS2 were ligated to the 3′‐end of sgRNA,and MCP was fused to the 5′‐end of cytosine deaminases.The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions(C‐T)at the target site.Different Cas9 variants,different cytosine deaminases and different copy numbers of MS2 were tested in this system,and the different versions of base editors were compared by editing efficiency and window.Results:In this study,dCas9,nCas9(D10A)and nCas9(H840A)were used.Among these 3 Cas9 variants,dCas9 exhibited higher base mutation efficiency.Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID.We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12.Disappointingly,the sgRNA‐12x MS2 did not improve the editing efficiency or increase the editing window.Conclusion:An optimal version of base editor based on the MS2 system,MS2‐BErAPOBEC1(sgRNA‐2x MS2,MCP‐rAPOBEC1 and dCas9),was obtained.This tool can simultaneously mutate multiple bases at the target site,providing a new approach for the study of genome functions.
基金supported by the National Key R&D Program(2016YFC0905901 to X.H.,2016YFA0503300 to X.G.)the NSFC(81771641 to X.G.)+1 种基金Fok Ying Tung Education Foundation(161037 to X.G.)Local Grants(17JC1420103 to X.H.,SKLRM-K201502 to X.G.)
文摘Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.