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. 2017 Sep 7;170(6):1224-1233.e15.
doi: 10.1016/j.cell.2017.07.037. Epub 2017 Aug 24.

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Free PMC article

A Broad-Spectrum Inhibitor of CRISPR-Cas9

Lucas B Harrington et al. Cell. .
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CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

Keywords: CRISPR; CRISPR-Cas9; Cas9; anti-CRISPR; gene editing.


Figure 1
Figure 1. AcrIIC1 inhibits diverse Cas9 orthologs while AcrIIC2 and AcrIIC3 are highly specific
(A) Unrooted phylogenetic tree of Cas9. Cas9 orthologs targeted by Acrs are indicated with circles at ends of branches (closed circles, Cas9 orthologs naturally targeted by an Acr; open circles, Cas9 orthologs which have been shown experimentally to be inhibited by an Acr but without naturally occurring AcrIIC1 orthologs). For branches containing multiple Acrs of a given type only one circle is shown for simplicity (phylogeny adapted form Burstein et al., 2017). (B) DNA cleavage assays conducted by various Cas9 orthologs in the presence of AcrIIC1, AcrIIC2 and AcrIIC3. (–Cas9, no Cas9 added; +Cas9, Cas9 and sgRNA added; Cje, Campylobacter jejuni; Nme, Neisseria meningitidis; Geo, Geobacillus stearothermophilus; Spy, Streptococcus pyogenes). (C) (Left) Cartoon depicting experiment to test inhibition of Cas9 orthologs by AcrIIC1 in HEK293 cells. (Right) T7E1 assay analyzing indels produced by CjeCas9 and NmeCas9 shows that CjeCas9 genome editing is inhibited by AcrIIC1Nme but not AcrIIC3Nme. See also Figure S1.
Figure 2
Figure 2. AcrIIC1 traps the DNA-bound Cas9 complex
(A) Cartoon of Cas9-mediated double-stranded DNA cleavage. Guide RNA (black) is duplexed to the DNA target strand (red), which is splayed from the DNA non-target strand (blue) adjacent to the PAM sequence (yellow). The HNH and RuvC nuclease domains (black triangles) cleave the target strand and non-target strand, respectively. (B) Radiolabeled cleavage assays conducted using GeoCas9 to measure AcrIIC1 inhibition of cleavage on the target and non-target strands. Cas9–sgRNA RNP was complexed with or without AcrIIC1 and added to radiolabeled target DNA duplex with each strand labeled separately. The lanes for a given condition correspond to increasing time (0-30min) from left to right. Black triangles indicate cleavage products. (C) Analysis of GeoCas9 binding and cleavage in the presence or absence of AcrIIC1 analyzed on a non-denaturing gel with the non-target strand labeled. GeoCas9 RNP concentration was varied in the absence or presence of excess AcrIIC1. The top band corresponds to GeoCas9 bound to the target DNA, the middle band is free DNA, and the lower band is cleaved DNA (concentration series correspond to 0, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512nM of GeoCas9 RNP). See also Figure S2.
Figure 3
Figure 3. AcrIIC1 binds to Cas9 HNH domain
(A) Domain schematics of GeoCas9 truncations and Cas9 chimeras, designed to identify the Cas9 binding interface of AcrIIC1. Constructs 1-10 were incubated with AcrIIC1, fractionated over an S200 size-exclusion column and analyzed by SDS-PAGE. Constructs that bound to AcrIIC1 are indicated with a (+) and constructs that showed no interaction are indicated with a (−). The chimeric Cas9 proteins (7-10) were generated by switching the HNH domains of a Cas9 that is not inhibited by AcrIIC1 (AnaCas9) and a Cas9 that is inhibited (GeoCas9). (B) Fractions from the S200 runs in Figure S3A were separated on a 4-20% SDS-PAGE gel. Numbers above the gel correspond to the construct or chimera numbers from Figure 2A. (C) Elution from an S75 size exclusion column of NmeCas9 HNH domain (purple), AcrIIC1 (orange) or the two incubated together with 2-fold excess AcrIIC1(red). See also Figure S3.
Figure 4
Figure 4. Structure of AcrIIC1 bound to the NmeCas9 HNH domain
(A) (Top) Cartoon depiction of NmeCas9 (grey) bound to a guide RNA (black.) The black outline of the HNH domain (purple) indicates the binding interface to AcrIIC1. (Bottom) Crystal structure of NmeCas9 HNH domain bound to AcrIIC1 (PDB:5VGB). Catalytic residues are depicted as sticks. (B) Occlusion of HNH active site residues (purple) through hydrogen bonding with AcrIIC1 (orange). HNH catalytic residues H588 and D587 form hydrogen bonds (black dotted line) with S78 and the backbone amine of C79 of AcrIIC1, respectively. 2mFo-DFc electron density map is shown for interacting residues and contoured at 1.8 σ. (C) Plaquing of E. coli phage Mu targeted by GeoCas9 in the presence of wild-type AcrIIC1 or the S78A AcrIIC1 mutant. Mutation of S78A results in nearly complete inactivation of AcrIIC1’s inhibitory effect on GeoCas9. (D) Binding interfaces of NmeCas9 HNH domain and AcrIIC1 show residue conservation. Conservation was calculated using multiple sequence alignments of AcrIIC1 orthologs and Cas9 HNH domains. Conserved residues are colored red (1, 100% sequence identity) and non-conserved residues are colored white (0). (E) Model of AcrIIC1 inhibiting cleavage of both target and non-target strands. NmeCas9 HNH domain (purple) was modeled into a “docked” position using dsDNA-bound SpyCas9 structure (PDB: 5F9R) as a reference for a homology model of NmeCas9. Placement of AcrIIC1 (orange) between the HNH domain and the target strand (red) prevents target cleavage and activation of the RuvC domain for non-target strand (dark blue) cleavage. See also Figure S4, S5 and Table S1.
Figure 5
Figure 5. AcrIIC3 blocks DNA binding and dimerizes Cas9
(A) Equilibrium binding measurements of NmeCas9 to dsDNA using fluorescence polarization in the presence (blue) or absence (black) of AcrIIC3. Measurements were made in triplicate and the mean +/− S. D. is shown. (B) Elution from a Superdex 200 10/300 size exclusion column for NmeCas9 (black), NmeCas9+AcrIIC1 (orange), and NmeCas9+AcrIIC3 (blue) showing a large shift in elution volume for NmeCas9-AcrIIC3, indicative of oligomerization. (C) SAXs data for fractions collected from samples in (C). (Left) pair-distance distribution function for NmeCas9 alone (black), with AcrIIC1 (orange) or with AcrIIC3 (blue), indicating increased particle size upon AcrIIC3 binding. (Rg, radius of gyration; Vc, volume of correlation; Dmax, maximum dimension.) (D) 2D class averages of NmeCas9-sgRNA monomers (left) and NmeCas9-sgRNA bound to AcrIIC3 (right). Scale bar is 10nm. See also Figure S2 and S6.
Figure 6
Figure 6. Model of AcrIIC1 and AcrIIC3 inhibition of Cas9
Cas9 assembles with its guide RNA to form the search complex. Phage encoded AcrIIC1 (orange) binds to Cas9, still allowing target dsDNA binding but occluding the HNH (purple) active site, and stopping cleavage of the target strand. AcrIIC1 also conformationally restricts HNH docking, stopping cleavage on the non-target strand. For AcrIIC3 (blue), Cas9’s target DNA binding is inhibited and Cas9 is caused to dimerize.

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