Shi, Ya-Jing, Duan, Min, Ding, Jun-Mei, Wang, Fan-Qi, Bi, Li-Li, Zhang, Cai-Xiang, Zhang, Yi-Zhou, Duan, Jun-Yi, Huang, An-Hui, Lei, Xin-Lin, Yin, Hao, and Zhang, Ying
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 N 4 CNNN and N 4 RNNA (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 near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing. [Display omitted] • Identification of a thermophilic AtCas9 with broad PAM CNNN and RNNA (R = A/G) • AtCas9 has a unique PAM-Cas interaction mechanism that can be regulated by DNA topology • AtCas9 shows near-PAMless cleavage in E. coli and effective base editing in 293T cells Shi et al. identified a thermophilic AtCas9, which has a relaxed PAM, and the PAM interaction can be robustly regulated by DNA topology. AtCas9 exhibits near-PAMless editing of supercoiled plasmids in E. coli and effective base-editing activity in mammalian cells. [ABSTRACT FROM AUTHOR]