After unsupervised three-dimensional (3D) classification of 840,000 particle images, refinement of a class containing 285,600 particles resulted in an EM reconstruction of the Sp圜as9-sgRNA-AcrIIA4 complex with an overall resolution of 3.9 Å (fig. Cryo-EM images were collected on a Krios microscope using zero-loss energy-filtered imaging and a K2 direct electron detector. To further elucidate the detailed molecular basis of AcrIIA4-mediated inhibition of Cas9 activity, we performed cryo–electron microscopy (cryo-EM) single-particle analysis on a Sp圜as9-sgRNA complex bound to AcrIIA4. ( C) Coomassie (CCB)– and ethidium bromide (EB)–stained polyacrylamide gel showing the comigration of AcrIIA4 with Cas9 in the presence of gRNA. Relevant peaks are indicated with arrowheads. ( B) Size exclusion chromatogram of Sp圜as9-sgRNA in the presence or absence of sgRNA after preincubation with AcrIIA4. Cas9-sgRNA complexed with AcrIIA4 is unable to bind to the target DNA. ( A) A cartoon depiction of Cas9 protein loaded with the sgRNA binding to AcrIIA4 (pink).
These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.ĪcrIIA4 binds to the Sp圜as9-sgRNA complex. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. A 3.9 Å resolution cryo–electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9–single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. Oligonucleotides used in this study.ĬRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target.
Data collection and model refinement statistics. Quantification of on- and off-target editing at HBB, as measured by TIDE analysis. Representative flow cytometry data used to create the graph shown in Fig. Western blot of AcrIIA4-3XFLAG expression.įig. Representative flow cytometry data used to create Fig. EMSA of Cas9-sgRNA binding to a target DNA with and without AcrIIA4.įig. EMSA of increasing concentrations of Cas9 binding to sgRNA in the absence or presence of AcrIIA4.įig.
Biological replicate data for BLI data shown in Fig. Model comparison between AcrIIA4-bound and DNA-bound Sp圜as9-sgRNA complexes.įig. Classification and refinement workflow.įig. Cryo-EM of Cas9 ribonucleoprotein particles.įig. Exposed region analysis of Sp圜as9 at AcrIIA4-free and AcrIIA4-bound states.įig. Gel filtration of Cas9 complexes with AcrIIA4.įig. Supplementary material for this article is available at įig.