Abstract
CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.
Highlights
CRISPR-Cas[9] technology has been widely used for genome engineering
A recent study revealed a second conformational sate, in which the HNH domain is closer to the cleavage site[11], the distance from the Cα atom of catalytic residue 840 to the attacked phosphorus is still more than 19 Å (HNH-state 2 in Supplementary Fig. 1c)
Compared with two known HNH states of SpCas[9] (Supplementary Fig. 1), this structure captures a distinct conformational state in which the HNH domain is nearest to the DNA cleavage site
Summary
CRISPR-Cas[9] technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas[9] binds to target DNA and cleaves the two DNA strands with HNH and RuvC nuclease domains. We report a 5.2 Å cryo-EM structure of Cas[9] in complex with sgRNA and target DNA This structure reveals a conformational state of Cas[9] in which the HNH domain is closest to the DNA cleavage site. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavageactivating architecture of Cas[9]. Compared with two known HNH states of SpCas[9] (Supplementary Fig. 1), this structure captures a distinct conformational state in which the HNH domain is nearest to the DNA cleavage site. This study provides mechanistic insights into the DNA cleavage by Cas[9], and will facilitate engineering efforts to improve the specificity and efficiency of the CRISPRCas[9] system
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