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Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein


CRISPR–Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA1,2,3,4,5. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells6. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing ‘off-switch’ tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

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Figure 1: AcrIIA2 and AcrIIA4 inhibit SpyCas9 activity by directly interacting with the sgRNA-bound SpyCas9.
Figure 2: Overall structure of AcrIIA4–SpyCas9–sgRNA.
Figure 3: Structural mechanism of AcrIIA4 recognition by SpyCas9.
Figure 4: AcrIIA4 and PAM sequence overlap when interacting with SpyCas9

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We thank J. He at Shanghai Synchrotron Radiation Facility (SSRF) for help with data collection. We thank J. Chai for critical reading of the manuscript. This research was funded by the National Natural Science Foundation of China grant no. 31422014, 31450001 and 31300605 to Z.H.

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Authors and Affiliations



D.D., M.G. and S.W. expressed, purified, characterized and crystallized the AcrIIA4–SpyCas9–sgRNA complex with the help of S.W., Z.X., J.Y. and Z.X. D.D., M.G., Y.Z. and Z.H. carried out crystallographic studies. D.D., M.G. and Z.H. prepared the figures. D.D. and M.G. performed in vitro transcription of sgRNA, in vitro dsDNA cleavage, GST pull-down, gel filtration, microscale thermophoresis and electrophoretic mobility shift assay experiments with the help of S.W., Z.X., J.Y. and Z.X. Z.H., D.D., M.G. and S.W. wrote the paper. All authors contributed to the manuscript preparation. Z.H. designed the experiments.

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Correspondence to Zhiwei Huang.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks S. Bailey, J. van der Oost and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 AcrIIA2 and AcrIIA4 specifically interact with sgRNA-bound SpyCas9.

Binding assays were carried out between the anti-CRISPR protein of AcrIIA2 or AcrIIA4 and the GST-tagged CRISPR protein of SpyCas9 or NmeCas9 in the presence or absence of cognate sgRNA. The purified GST–SpyCas9 or GST–NmeCas9 protein was first bound to glutathione sepharose beads in the presence or absence of sgRNA, and then the beads were incubated them with AcrIIA2 or AcrIIA4 protein. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. Data shown are the representative of three replicates.

Extended Data Figure 2 Alignment of Cas9 protein sequences.

Multiple sequence alignment of the amino acid sequences of type II-A Cas9 proteins from Streptococcus pyogenes (GI 15675041), Listeria monocytogenes J0161 (GI 345535315), Listeria innocua Clip11262 (GI 16414891), and type II-C Cas9 proteins of Neisseria meningitidis (GI 518572566), Pasteurella multocida subsp. multocida str. Pm70 (GI 218767588), aligned using MUSCLE. Residues with more than 70% similarity are shown in red and boxed in blue. Residues involved in interaction with AcrIIA4 are indicated.

Extended Data Figure 3 Structural comparison of SpyCas9–sgRNA–DNA and SpyCas9–sgRNA–AcrIIA4.

a, Structural superimposition of SpyCas9–sgRNA–DNA (PDB code, 4UN3) and SpyCas9–sgRNA–AcrIIA4. b, GST pull-down assays to verify the structural determinants for preferential binding of AcrIIA4 to SpyCas9. Wild-type or mutant GST-fused AcrIIA4 proteins were first bound to glutathione sepharose beads and incubated with sgRNA-preloaded SpyCas9 (or mutant) protein as indicated. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. c, Enzymatic activity assays to verify structural determinants for specific AcrIIA4–SpyCas9 interaction. The assays were performed as described in Fig. 1b. Data shown are representative of three independent experiments. d, AcrIIA2 and AcrIIA4 compete with PAM-containing dsDNA for binding to the SpyCas9–sgRNA. sgRNA-preloaded GST–SpyCas9 protein was first mixed with AcrIIA2 or AcrIIA4 at 4 °C and incubated for 15 min, followed by addition of PAM-containing dsDNA into the reaction mixtures. After 15 min incubation, the reactions were stopped by adding loading buffer for denaturing gel and the reaction mixtures were loaded onto glutathione sepharose beads and incubated for 15 min. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. Data shown are representative of three independent experiments. e, Electrophoretic mobility shift assay results showing that AcrIIA2 and AcrIIA4 compete with PAM-containing dsDNA for binding to the SpyCas9–sgRNA. The fluorophore-labelled dsDNA and AcrIIA2 (upper panel) or AcrIIA4 (lower panel) were added to sgRNA-preloaded inactive SpyCas9(D10A/H840A) simultaneously. Molar ratios of SpyCas9–anti-CRISPR protein are shown at the top of each lane. The reaction mixtures were run on 6% native polyacrylamide gels and visualized by fluorescence imaging (800 nm). Data shown are representative of three independent experiments.

Extended Data Figure 4 Structural comparison of SpyCas9–sgRNA and SpyCas9.

Structural superimposition of SpyCas9–sgRNA (PDB code, 4ZT0) and SpyCas9 (PDB code, 4CMP). The interface of AcrIIA4 and SpyCas9 is circled in black dashed line.

Extended Data Table 1 Data collection, phasing and refinement statistics
Extended Data Table 2 Nucleic acid sequences used in the study

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Dong, D., Guo, M., Wang, S. et al. Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein. Nature 546, 436–439 (2017).

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