文献类型：期刊文献

英文题名：Genome editing with natural and engineered CjCas9 orthologs

作者：Gao, Siqi[1] Wang, Yao[1] Qi, Tao[1] Wei, Jingjing[1] Hu, Ziying[1] Liu, Jingtong[1] Sun, Shuna[2] Liu, Huihui[3] Wang, Yongming[1]

第一作者：Gao, Siqi

通信作者：Wang, YM[1];Sun, SA[2];Liu, HH[3]

机构：[1]Fudan Univ, Zhongshan Hosp, Sch Life Sci, State Key Lab Genet Engn, Shanghai 200438, Peoples R China;[2]Fudan Univ, Childrens Hosp, Natl Childrens Med Ctr, Shanghai 201102, Peoples R China;[3]Chinese Acad Forestry, Expt Ctr Forestry North China, Beijing 102300, Peoples R China

年份：2023

卷号：31

期号：4

起止页码：1177-1187

外文期刊名：MOLECULAR THERAPY

收录：;Scopus(收录号：2-s2.0-85150420001);WOS:【SCI-EXPANDED(收录号:WOS:000982603300001)】；

基金：This work was supported by grants from the National Key Research and Development Program of China (2021YFC2701103 and 2021YFA0910602); the National Natural Science Foundation of China (82070258, 31700571, and 82270313); Open Research Fund of State Key Laboratory of Genetic Engineering of Fudan University (no. SKLGE-2104); Science and Technology Research Program of Shanghai (19DZ2282100); the Natural Science Fund of Shanghai Sci-ence and Technology Commission (19ZR1406300); and Shanghai 2022 "Science and Technology Innovation Action Plan" Medical Innovation Research Project (22Y11909300).

语种：英文

外文关键词：chimeric Cas9; CjCas9; compact Cas9; Compact Cas9; CRISPR/Cas9; high-fidelity; Hsp1-Hsp2Cas9

摘要：CjCas9 is one of the smallest CRISPR-associated (Cas9) nu-cleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adja-cent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.