Research ArticleBIOCHEMISTRY

Disabling Cas9 by an anti-CRISPR DNA mimic

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Science Advances  12 Jul 2017:
Vol. 3, no. 7, e1701620
DOI: 10.1126/sciadv.1701620
  • Fig. 1 AcrIIA4 binds to the SpyCas9-sgRNA complex.

    (A) A cartoon depiction of Cas9 protein loaded with the sgRNA binding to AcrIIA4 (pink). Cas9-sgRNA complexed with AcrIIA4 is unable to bind to the target DNA. (B) Size exclusion chromatogram of SpyCas9-sgRNA in the presence or absence of sgRNA after preincubation with AcrIIA4. Relevant peaks are indicated with arrowheads. (C) Coomassie (CCB)– and ethidium bromide (EB)–stained polyacrylamide gel showing the comigration of AcrIIA4 with Cas9 in the presence of gRNA.

  • Fig. 2 Architecture of the SpyCas9-sgRNA in complex with AcrIIA4.

    (A) Cryo-EM reconstruction of the AcrII4-bound SpyCas9. The electron density map was contoured at high-threshold levels showing distinct features for each subunit. (B) Representative cryo-EM density for AcrIIA4 with the refined model superimposed. (C) The atomic model of SpyCas9-sgRNA-ArcrIIA4. AcrIIA4 (red) and sgRNA (orange) are shown in a ribbon diagram. (D) Surface representation showing AcrIIA4 binding to the PAM-recognition cleft. (E) Superposition with Cas9–sgRNA–dsDNA (double-stranded DNA) structure (PDB ID: 5F9R). For clarity, Cas9 is omitted except the HNH domain. Target and nontarget DNA strand is colored purple and beige, respectively. (F) Electrostatic surface potential of AcrIIA4 showing that AcrIIA4 fits perfectly into the major groove of PAM duplex and that its surface acts as a dsDNA mimic. The inset shows that the PAM recognition residues (R1333 and R1334) are largely buried in an acidic pocket within AcrIIA4.

  • Fig. 3 AcrIIA4 inhibits DNA cleavage in vitro.

    (A) Time-course cleavage assay using linearized plasmid template containing a 20–base pair λ1 DNA target sequence and a 5′-TGG-3′ PAM motif showing that AcrIIA4 inhibits SpyCas9-mediated endonuclease activity. (B) AcrIIA4 inhibits SpyCas9 cleavage of radiolabeled target DNA in vitro. SpyCas9–crRNA (CRISPR RNA)–tracrRNA (trans-activating crRNA) complex (10 nM) was preincubated with increasing concentrations (0 to 100 nM) of AcrIIA4. Substoichiometric synthetic oligonucleotide duplexes (2.5 nM) bearing a radiolabel at the 5′ end of the complementary strand were introduced for 6-min cleavage reactions. Reactions were resolved by denaturing polyacrylamide gel electrophoresis and visualized by phosphorimaging. (C) AcrIIA4 inhibits dCas9-sgRNA binding to a DNA target but does not affect target release, as measured by BLI. Preincubation in increasing concentrations of AcrIIA4 with dCas9-sgRNA reduces the on-rate of association with a DNA target relative to no inhibitor (blue). Maximal inhibition is identical to dCas9-sgRNA added to target DNA with a PAM mutation (pink). (D) Addition of increasing concentrations of AcrIIA4 with the preformed dCas9-sgRNA-DNA complex has no effect on the off-rate of dissociation.

  • Fig. 4 Timed delivery of AcrIIA4 differentially inhibits on- and off-target genome editing in human cells.

    (A) Simultaneous delivery of Cas9 RNP and AcrIIA4 inhibits Cas9-mediated gene targeting in human cells. K562 cells with a chromosomally integrated BFP (BFP-K562) were nucleofected with Cas9 RNP and AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:0.5, 1:1, 1:2, 1:3, and 1:5] or plasmid (0.7 and 2.8 μg). Nonhomologous end joining (NHEJ) frequencies are quantified by the loss of BFP expression in BFP-K562 cells 96 hours after nucleofection via flow cytometry. Data are presented as means ± SEM from at least two biological replicates. (B) Administration of AcrIIA4 before Cas9 RNP completely inhibits Cas9-mediated gene targeting. BFP-K562 cells were nucleofected with AcrIIA4 plasmid (0.7 μg) 24 hours before Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. (C) Delivery of AcrIIA4 after introduction of Cas9 RNP yields intermediate inhibition of Cas9 activity. BFP-K562 cells were nucleofected with AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) 6 hours after Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. (D) Proper timing of AcrIIA4 delivery diminishes off-target editing events while largely retaining on-target editing. K562 cells were nucleofected with either HBB or EMX1 targeting Cas9 RNP 6 hours before AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) delivery (18). Representative T7 endonuclease I assay for visualization of HBB on- and off-target editing. Bottom: Inhibition of editing by AcrIIA4 at on- and off-target sites of HBB and VEGFA, as measured by amplicon sequencing.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1701620/DC1

    fig. S1. Gel filtration of Cas9 complexes with AcrIIA4.

    fig. S2. Exposed region analysis of SpyCas9 at AcrIIA4-free and AcrIIA4-bound states.

    fig. S3. Cryo-EM of Cas9 ribonucleoprotein particles.

    fig. S4. Classification and refinement workflow.

    fig. S5. Atomic modeling.

    fig. S6. Model comparison between AcrIIA4-bound and DNA-bound SpyCas9-sgRNA complexes.

    fig. S7. Biological replicate data for BLI data shown in Fig. 3C.

    fig. S8. EMSA of increasing concentrations of Cas9 binding to sgRNA in the absence or presence of AcrIIA4.

    fig. S9. EMSA of Cas9-sgRNA binding to a target DNA with and without AcrIIA4.

    fig. S10. Representative flow cytometry data used to create Fig. 4A.

    fig. S11. Western blot of AcrIIA4-3XFLAG expression.

    fig. S12. Representative flow cytometry data used to create the graph shown in Fig. 4B.

    fig. S13. Representative flow cytometry data used to create the graph shown in Fig. 4C.

    fig. S14. Quantification of on- and off-target editing at HBB, as measured by TIDE analysis.

    table S1. Data collection and model refinement statistics.

    table S2. Oligonucleotides used in this study.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Gel filtration of Cas9 complexes with AcrIIA4.
    • fig. S2. Exposed region analysis of SpyCas9 at AcrIIA4-free and AcrIIA4-bound states.
    • fig. S3. Cryo-EM of Cas9 ribonucleoprotein particles.
    • fig. S4. Classification and refinement workflow.
    • fig. S5. Atomic modeling.
    • fig. S6. Model comparison between AcrIIA4-bound and DNA-bound SpyCas9-sgRNA complexes.
    • fig. S7. Biological replicate data for BLI data shown in Fig. 3C.
    • fig. S8. EMSA of increasing concentrations of Cas9 binding to sgRNA in the absence or presence of AcrIIA4.
    • fig. S9. EMSA of Cas9-sgRNA binding to a target DNA with and without AcrIIA4.
    • fig. S10. Representative flow cytometry data used to create Fig. 4A.
    • fig. S11. Western blot of AcrIIA4-3XFLAG expression.
    • fig. S12. Representative flow cytometry data used to create the graph shown in Fig. 4B.
    • fig. S13. Representative flow cytometry data used to create the graph shown in Fig. 4C.
    • fig. S14. Quantification of on- and off-target editing at HBB, as measured by TIDE analysis.
    • table S1. Data collection and model refinement statistics.
    • table S2. Oligonucleotides used in this study.

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