Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing  

作　　者：Zhi-Xue Yang Ya-Wen Fu Juan-Juan Zhao Feng Zhang Si-Ang Li Mei Zhao Wei Wen Lei Zhang Tao Cheng Jian-Ping Zhang Xiao-Bing Zhang 

机构地区：[1]State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China [2]Center for Stem Cell Medicine,Chinese Academy of Medical Sciences,Tianjin 300020,China [3]Department of Stem Cell&Regenerative Medicine,Peking Union Medical College,Tianjin 300020,China

出　　处：《Genomics, Proteomics & Bioinformatics》2023年第6期1206-1220,共15页基因组蛋白质组与生物信息学报（英文版）

基　　金：supported by the National Natural Science Foundation of China(Grant Nos.82070115,81770198,81870149,81970121,and 8142100);the National Key R&D Program of China(Grant Nos.2019YFA0110803,2019YFA0110802,2019YFA0110204,and 2016YFA0100600);the Tianjin Municipal Science and Technology Commission Grant(Grant No.19JCZDJC33000);the CAMS Innovation Fund for Medical Sciences(Grant Nos.2017-I2M-2-001,2017-I2M-B&R-04,and 2019-I2M-1-006).

摘　　要：A series of clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)systems have been engineered for genome editing.The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus.However,a comparison of their detailed gene editing outcomes is still lacking.By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells(iPSCs)and K562 cells,we found that SaCas9 could edit the genome with greater efficiencies than SpCas9.We also compared the effects of spacer lengths of single-guide RNAs(sgRNAs;18–21 nt for SpCas9 and 19–23 nt for SaCas9)and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9,respectively.However,the optimal spacer length for a particular sgRNA was 18–21 nt for SpCas9 and 21–22 nt for SaCas9.Furthermore,SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining(NHEJ)+1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif(PAM),indicating a characteristic of a staggered cut.Accordingly,editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide(dsODN)insertion or homology-directed repair(HDR)-mediated adeno-associated virus serotype 6(AAV6)donor knock-in.Finally,GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9.Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

关 键 词：SpCas9 SaCas9 Spacer length Indel pattern Knock-in efficiency Off-target 

分 类 号：Q943.2[生物学—植物学]