Conformational control of DNA target cleavage by CRISPR–Cas9

By S. H. Sternberg, B. LaFrance, M. Kaplan, Jennifer A. Doudna. 2015

569 citations recorded by the source corpus.

Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Together with single-guide RNAs, Cas9 also functions as a powerful genome engineering tool in plants and animals, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition and RNA–DNA base-pairing that serves as a final specificity checkpoint before DNA double-strand break formation.

Subfields: Molecular Biology

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, neutral): “2 and Extended Data Figs 5–7) may reflect more extensive requirements for DNA cleavage than DNA binding 27,32 , or differences in the chromatin state of plasmid (Fig.”
Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. (2016, unknown): “FRET assays show that the HNH domain swings into a catalytically competent position only upon binding to a cognate double-stranded DNA substrate, underscoring the multiple steps of conformational control of Cas9-catalyzed DNA cleavage (Sternberg et al., 2015).”
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems (2016, background): “This R-loop triggers a conformational change of the two nuclease domains (HNH and RuvC) of Cas9, which adopt an active state that allows for the completion of interference by cleavage of both target strands [120, 122].”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “The extent of this HNH domain conformational change, monitored by intramolecular Förster resonance energy transfer (FRET) assays, is sensitive to mismatches within the guide RNA–target DNA base-pairing region (86).”
The Biology of CRISPR-Cas: Backward and Forward. (2018, unknown): “This allosteric communication be-tween the nuclease domains results in concerted cleavage of the target strand by the HNH domain and the non-target strand by the RuvC domain (Jinek et al., 2012; Sternberg et al., 2015).”
Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, unknown): “4 nt of additional matches to activate the HNH catalytic domain (Sternberg et al., 2015).”