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

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

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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 out-comes 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 nonhomolo-gous end-joining(NHEJ)+1 insertion at the fourth nucleotide upstream of the protospacer adja-cent motif(PAM),indicating a characteristic of a staggered cut.Accordingly,editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide(dsODN)inser-tion 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.

SpCas9SaCas9Spacer lengthIndel patternKnock-in efficiencyOff-target

Zhixue Yang、Yawen Fu、Juanjuan Zhao、Feng Zhang、Siang Li、Mei Zhao、Wei Wen、Lei Zhang、Tao Cheng、Jianping Zhang、Xiaobing Zhang

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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

Center for Stem Cell Medicine,Chinese Academy of Medical Sciences,Tianjin 300020,China

Department of Stem Cell & Regenerative Medicine,Peking Union Medical College,Tianjin 300020,China

National Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Natural Science Foundation of ChinaNational Key R&D Program of ChinaNational Key R&D Program of ChinaNational Key R&D Program of ChinaNational Key R&D Program of ChinaTianjin Municipal Science and Technology Commission GrantCAMS Innovation Fund for Medical SciencesCAMS Innovation Fund for Medical SciencesCAMS Innovation Fund for Medical Sciences

8207011581770198818701498197012181421002019YFA01108032019YFA01108022019YFA01102042016YFA010060019JCZDJC330002017-I2M-2-0012017-I2M-B&R-042019-I2M-1-006

2023

10.1016/j.gpb.2022.12.003

基因组蛋白质组与生物信息学报(英文版)
中国科学院北京基因组研究所

基因组蛋白质组与生物信息学报(英文版)

CSTPCDCSCD
影响因子：0.495
ISSN：1672-0229
年,卷(期)：2023.6(6)
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