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Article Dans Une Revue Science Advances Année : 2019

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

Résumé

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a singlemolecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.
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Dates et versions

hal-03081006 , version 1 (18-12-2020)

Identifiants

Citer

Qian Zhang, Fengcai Wen, Siqi Zhang, Jiachuan Jin, Lulu Bi, et al.. The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation. Science Advances , 2019, 5 (11), ⟨10.1126/sciadv.aaw9807⟩. ⟨hal-03081006⟩
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