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

Abstract

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 RNA. 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 cells. 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.

A Broad-Spectrum Inhibitor of CRISPR-Cas9

“…However, when AcrIIC1 was included in the reaction, Cas9 did not cleave the target DNA even though DNA binding was unaffected. This remarkable mechanism is distinct from the recently studied AcrIIA2 and AcrIIA4 anti-CRISPR proteins, which function as inhibitors of DNA binding by SpyCas9 (Dong et al, 2017; Shin et al, 2017). The unique ability of AcrIIC1 to trap Cas9 on its DNA target in a catalytically inactivate state effectively transforms the wild-type Cas9 into its catalytically inactive variant dCas9 (Jinek et al, 2012).…”

“…In contrast, the 116-amino acid AcrIIC3 binds specifically to the NmeCas9 enzyme to trigger dimerization and prevent DNA binding. Both of these mechanisms are different from that of the anti-CRISPR protein AcrIIA4, which acts as a DNA mimetic that prevents DNA binding by occupying the PAM-recognition site within a small subset of related type IIA Cas9 orthologs (Dong et al, 2017; Shin et al, 2017). …”

“…The first and most common mechanism is to target the CRISPR surveillance complex by disrupting DNA binding (AcrIIC3, AcrIIA4, AcrF1, AcrF2; (Bondy-Denomy et al, 2015; Chowdhury et al, 2017; Dong et al, 2017; Pawluk et al, 2016b; Rauch et al, 2017; Shin et al, 2017). The second is to target nucleases or nuclease domains, thereby allowing DNA binding but not cleavage (AcrIIC1, AcrF3; (Bondy-Denomy et al, 2015; Wang et al, 2016).…”

“…AcrIIC3, by contrast, inhibits only a single Cas9 ortholog by blocking DNA binding. AcrIIC3 also causes Cas9 to dimerize, possibly contributing to its ability to interfere with target recognition and suggesting a mechanism distinct from that observed for AcrIIA4 (Dong et al, 2017; Shin et al, 2017). Together, AcrIIC1 and AcrIIC3 enable either broad-spectrum or selective inhibition of Cas9 orthologs respectively.…”

Inhibition of CRISPR-Cas systems by mobile genetic elements

“…Finally, both AcrIIA2 Lmo and AcrIIA4 Lmo were shown to inhibit transcriptional repression by promoter-directed dSpyCas9 in E. coli [41], indicating that both Acrs prevent stable, sgRNA-guided DNA binding by dSpyCas9. Three recent reports describe the structure of AcrIIA4 Lmo in complex with sgRNA-loaded SpyCas9, revealing a DNA-mimicking inhibitor that binds to the PAM-interacting region of the SpyCas9/sgRNA complex [44–46], reminiscent of the inhibitory logic exhibited by the type I inhibitor AcrF2 (see above). In agreement with the functional tests of Rauch et al [41], AcrIIA4 Lmo binding prevents the engagement of sgRNA-complementary target DNA by SpyCas9.…”