Engineered Staphylococcus auricularis Cas9 with high-fidelity.

Autor: Wei N; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Shang L; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Liu J; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Wang M; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Liu Y; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Zhu C; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China., Fei C; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Zhang L; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Yang F; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China., Gu F; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.; Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai, China.
Jazyk: angličtina
Zdroj: FASEB journal : official publication of the Federation of American Societies for Experimental Biology [FASEB J] 2023 Aug; Vol. 37 (8), pp. e23060.
DOI: 10.1096/fj.202202132RR
Abstrakt: CRISPR-Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off-target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5'-NNGG-3' protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced-fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5'-NNGG-3' than SaCas9 of 5'-NNGRRT-3' PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9-HF1, harboring the N269D mutation; SauriCas9-HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE-seq. At certain sites, reduced off-target effects (approximately 61.6- and 111.9-fold improvements) of SauriCas9-HF2 compared with wild-type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9-HF1 and SauriCas9-HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.
(© 2023 Federation of American Societies for Experimental Biology.)
Databáze: MEDLINE
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