The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation
Open Access
- 1 November 2019
- journal article
- research article
- Published by American Association for the Advancement of Science (AAAS) in Science Advances
- Vol. 5 (11), eaaw9807
- https://doi.org/10.1126/sciadv.aaw9807
Abstract
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 single-molecule 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.Funding Information
- Ministry of Science and Technology of the People’s Republic of China (2016YFA0500902)
This publication has 41 references indexed in Scilit:
- DNA interrogation by the CRISPR RNA-guided endonuclease Cas9Nature, 2014
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- Single-molecule analysis of DNA replication in Xenopus egg extractsMethods, 2012
- ATP-induced helicase slippage reveals highly coordinated subunitsNature, 2011
- CRISPR interference: RNA-directed adaptive immunity in bacteria and archaeaNature Reviews Genetics, 2010
- Short motif sequences determine the targets of the prokaryotic CRISPR defence systemMicrobiology, 2009
- Characterization and Mutational Analysis of the RecQ Core of the Bloom Syndrome ProteinJournal of Molecular Biology, 2003
- Decreased Resistance to Bacterial Infection and Granulocyte Defects in IAP-Deficient MiceScience, 1996
- Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA MoleculesScience, 1996
- Entropic Elasticity of λ-Phage DNAScience, 1994