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DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9

Mol Cell. 2022-11; 
Ya-Jing Shi , Min Duan , Jun-Mei Ding , Fan-Qi Wang , Li-Li Bi , Cai-Xiang Zhang , Yi-Zhou Zhang , Jun-Yi Duan , An-Hui Huang , Xin-Lin Lei , Hao Yin , Ying Zhang
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Bacterial Expression DH5-alpha or MG1655 electrocompetent E. coli cells (Weidi Biotechnology) were transformed with the PAM library plasmids(GenScript) or target plasmids. 6xHis tagged AtCas9 gene was synthesized and cloned into pET28a vector (GenScript). Get A Quote
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摘要

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed ne... More

关键词

AtCas9; CRISPR/Cas9; DNA topology; PAM; PAM-Cas interaction; Type II-C Cas9; base editing; near-PAMless; thermophilic cas9.
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