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A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules

Abstract

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

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Fig. 1: Development of the CAA.
Fig. 2: Generalization of the CAA to different Cas systems.
Fig. 3: High-throughput optimization and screening with the CAA.
Fig. 4: Cellular validation of BRD7586.
Fig. 5: Structure–activity relationship studies with BRD7586.
Fig. 6: Biochemical binding studies of BRD7586.
Fig. 7: BRD7586 mechanism-of-action studies.

Data availability

Data generated in this study are provided in the manuscript, Supplementary Information and Source Data. Plasmids from Addgene (plasmids 43861, https://www.addgene.org/43861; 47511, https://www.addgene.org/47511; and 42230, https://www.addgene.org/42230) were used in this study. Structural information from Protein Data Bank (ID: 5F9R) was used in this study. High-throughput sequencing data have been deposited at the NCBI Sequence Read Archive database under accession number PRJNA862731. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

No code or algorithm was generated in this study.

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Acknowledgements

This work was supported by the Burroughs Wellcome Fund (Career Award at the Scientific Interface to A.C.), DARPA (N66001-17-2-4055 to A.C.) and NIH (R01GM132825 and R01GM137606 to A.C.). We thank J. K. Joung for providing U2OS.eGFP.PEST cells and H. S. Malik for providing AcrIIA11 protein expression plasmid.

Author information

Authors and Affiliations

Authors

Contributions

D.L., Q.Z., K.J.C., B.K.L., M.L., P.K., V.S. and A.C. planned the research. D.L., Q.Z., K.J.C., B.K.L., M.L., P.K., V.S., Y.A., C.M.W. and A.C. designed the experiments. D.L., Q.Z., K.J.C., B.K.L., M.L., P.K., V.S., R.P., S.K.C., S.A.G., B.M., S.L., Y.A. and M.F.M. performed the experiments. D.L., Q.Z., K.J.C., B.K.L., M.L., P.K., V.S., R.P., S.K.C., S.A.G., B.M., S.L., Y.A., D.B.T., H.K.K.S., M.F,M., V.D., P.A.C., B.K.W., C.M.W, G.M.C. and A.C. analysed the data. D.L., Q.Z., K.J.C., B.K.L., M.L., P.K., V.S. and A.C. wrote the manuscript. A.C. supervised the research. R.P. and S.K.C. contributed equally to this work.

Corresponding author

Correspondence to Amit Choudhary.

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Competing interests

Broad Institute has filed a patent application including work described herein (US Provisional Patent Application Number 63/393,788; inventors: A.C., D.L., Q.Z., K.J.C., B.K.L., M.L., P.K. and V.S.). The remaining authors declare no competing interests.

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Nature Cell Biology thanks Keith T. Gagnon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Validation of the CAA.

(a) Schematic of the differently labelled PAM fluorescence polarization substrates. (b) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with SaCas9 at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 3′ end. SaCas9 showed specificity for the 12-PAM substrate that decreased with increasing amounts of unlabelled competitor. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No SaCas9’, p = 0.061 with the 0-PAM DNA and p = 1.1 × 10−4 with the 12-PAM DNA (unpaired t-test, two-tailed). (c) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with FnCas12a at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 3′ end. FnCas12a showed no specificity dependent upon the presence of PAM-binding sites. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No FnCas12a’, p = 3.5 × 10−5 with the 0-PAM and p = 7.4 × 10−7 with the 12-PAM DNA (unpaired t-test, two-tailed). (d) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with FnCas12a at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 5′ end. FnCas12a showed no specificity dependent upon the presence of PAM-binding sites. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No FnCas12a’, p = 6.2 × 10−4 with the 0-PAM and p = 6.2 × 10−5 with the 12-PAM DNA (unpaired t-test, two-tailed). (e) Inhibition of SpCas9 by AcrIIA11 monitored by the CAA. Error bars represent mean ± SD from 4 independent replicates. For 10 µM AcrIIA11 compared to buffer only, p = 1.6 × 10−7 (unpaired t-test, two-tailed). (f) The fluorescence of the SaCas9-specific substrate is not quenched in the presence of quencher unless the duplex is disrupted by cleavage via an active SaCas9:gRNA complex. A single DNA strand containing the fluorophore (SS-DNA) can be completely quenched in the absence of an unlabelled complementary strand. Error bars represent mean ± SD from 4 independent replicates. For SaCas9:gRNA (4th bar) compared to SaCas9 only (3rd bar). p = 1.1 × 10−8 (unpaired t-test, two-tailed).

Source data

Extended Data Fig. 2 Generalization of the CAA.

(a) Demonstration of FnCas12a CAA. The fluorescence of the FnCas12a-specific substrate labelled on the non-targeting strand (NTS) is not quenched in the presence of quencher unless the duplex is disrupted by cleavage via an active FnCas12a:gRNA complex. A single DNA strand containing the fluorophore (SS-DNA) can be completely quenched in the absence of an unlabelled complementary strand. Error bars represent mean ± SD from 4 independent replicates. For FnCas12a:gRNA (4th bar) compared to FnCas12a only (3rd bar), p = 1.5 × 10−7 (unpaired t-test, two-tailed). (b) Demonstration of FnCas12a CAA. The fluorescence of the FnCas12a-specific substrate labelled on the targeting strand (TS) is quenched poorly even in the presence of active FnCas12a:gRNA complex. Error bars represent mean ± SD from 4 independent replicates. For FnCas12a:gRNA (4th bar) compared to FnCas12a only (3rd bar), p = 3.3 × 10−5 (unpaired t-test, two-tailed). (c) Gel-monitored cleavage of FAM-labelled oligos (20 nM) by AsCas12a (100 nM), LbCas12a (100 nM), and FnCas12a (100 nM) in a PAM-dependent manner.

Source data

Extended Data Fig. 3 Biochemical validation of BRD7586.

(a) Hit compounds (Z score > 3σ) identified in both the primary screen and secondary screens. Compounds were compared to BRD0539, a compound previously identified as an SpCas9 DNA-binding inhibitor. (b) Dose-dependent inhibition of SpCas9 by BRD7586 in the CAA. Error bars represent mean ± SD from 3 independent replicates. For 30 µM of BRD7586 compared to DMSO, p = 0.020 (unpaired t-test, two-tailed). (c) Gel electrophoresis-based monitoring of dose-dependent inhibition of SpCas9 by BRD7586 from in vitro DNA cleavage assay. Images from 4 independent replicates are shown. (d) Quantification of the inhibition from the above in vitro DNA cleavage assay. Error bars represent mean ± SD from 4 biological replicates. For 40 µM (6th bar) compared to DMSO (1st bar), p = 1.3 × 10−7 (unpaired t-test, two-tailed).

Source data

Extended Data Fig. 4 Validation of BRD7586 in cells.

(a, b) T7E1 assay for detecting indels at the eGFP gene. U2OS.eGFP.PEST cells were nucleofected with (a) plasmid or (b) RNP, and incubated with BRD7586 for 24 h. Blue arrowheads indicate uncleaved DNA and black arrowheads indicate cleaved DNA. (c) Inhibition of SpCas9 by BRD0539 and BRD7586 in the eGFP disruption assay using plasmid delivery method (left) and RNP delivery method (right) (U2OS.eGFP.PEST cells, 24 h). Error bars represent mean ± SD from 3 (5th bar in plasmid delivery) or 4 (1st to 4th bars in plasmid delivery and all bars in RNP delivery) independent replicates. For the plasmid-based assay, p = 3.5 × 10−7 for BRD0539 and p = 1.2 × 10−5 for BRD7586 at 15 µM compared to DMSO. For the RNP-based assay, p = 4.1 × 10−4 for BRD0539 and p = 3.6 × 10−6 for BRD7586 at 15 µM compared to DMSO (unpaired t-test, two-tailed). (d) Inhibition of SpCas9 by BRD0539 and BRD7568 in the HiBiT knock-in assay using plasmid delivery method (left) and RNP delivery method (right) in HEK293T cells (48 h). Error bars represent mean ± SD from 3 independent replicates for the plasmid delivery. Data represents mean from 2 independent replicates for the RNP delivery. For the plasmid delivery, p = 7.8 × 10−4 for BRD0539 at 15 µM compared to DMSO, and p = 4.9 × 10−5 for BRD7586 at 15 µM compared to DMSO (unpaired t-test, two-tailed).

Source data

Extended Data Fig. 5 Counter assays to validate BRD7586 in cells.

(a, b) Immunoblotting analysis of SpCas9 expression in (a) HEK293T cells (data represent mean from 2 independent experiments) or (b) U2OS.eGFP.PEST cells (error bars represent mean ± SD from 3 independent experiments) transfected with SpCas9 plasmid and incubated with BRD7586 for 24 h. For BRD7586 at 20 µM compared to DMSO in U2OS.eGFP.PEST cells, p = 0.49 (unpaired t-test, two-tailed). (c) Changes in the fluorescence intensity from U2OS.eGFP.PEST cells in the presence of BRD7586. Cells were treated with the compound for 24 h, and the fluorescence intensity was measured to calculate the fraction of eGFP-positive populations. Means from 3 independent experiments are shown. Due to the low SD, error bars cannot be shown. For BRD7586 at 20 µM compared to DMSO, p = 0.14 (unpaired t-test, two-tailed). (d) Dose-dependent inhibition of SpCas9 or LbCas12a by BRD7586 in the eGFP disruption assay using RNP delivery methods (U2OS.eGFP.PEST cells, 24 h). Error bars represent mean ± SD from 3 independent experiments. For SpCas9, p = 8.3 × 10−5 with BRD7586 at 15 µM compared to DMSO. For LbCas12a, p = 0.023 with BRD7586 at 15 µM compared to DMSO (unpaired t-test, two-tailed).

Source data

Extended Data Fig. 6 Validation of the binding between BRD7586 and SpCas9.

(a) Structure of Biotin-BRD7586 and Biotin-PEG3-Azide control. (b) Control BLI binding plot for Biotin-PEG3-azide and SpCas9:gRNA complex. BLI experiment was performed using 1 µM of Biotin-PEG3-azide on streptavidin sensors followed by association with different concentrations of SpCas9:gRNA complex and subsequent dissociation. BLI signal of biotin-BRD7586 and 1 µM SpCas9:gRNA complex is marked in red for comparison.

Extended Data Fig. 7 Mechanism-of-action studies of BRD7586.

(a) T7E1 assay for measuring the activity of Diazirine-BRD7586. U2OS.eGFP.PEST cells were nucleofected with Cas9 plasmid and eGFP-targeting plasmid, and the cells were incubated with the compounds for 24 h. Blue arrowheads indicate uncleaved DNA while black arrowheads indicate cleaved DNA from the T7E1 reaction. (b) Workflow of the chemoproteomics experiments using the diazirine-based photo-crosslinking probe to identify binding sites of BRD7586 on SpCas9. (c) Chemical structure of the acid-cleavable and isotope-coded biotin-azide used for chemoproteomics experiments. (d) Structure of the peptide-compound conjugates to be detected from the mass spectrometry. Note the 3:1 ratio of the isotope tag that allows reliable identification of the conjugates. (E-F) Examples of the mass spectra obtained from the chemoproteomics experiments. For the detailed information of identified peptides, see Supplementary Table 8. The isotope patterns are shown in MS1 spectra. The probe-conjugated residues are shown as green labels in MS2 spectra. (g) Proposed binding pocket of BRD7586 on SpCas9. BRD7586 was docked to SpCas9 at the HNH-nuclease and helical recognition domains.

Source data

Extended Data Fig. 8 Mechanism of action studies of BRD7586.

(a) Structure of BRD7586 and F2537-0908. (b) Activity of BRD7586 and F2537-0908 in the eGFP disruption assay. Results from 2 independent experiments are shown. (c) Activity of BRD7586 and F2537-0908 in the HiBiT knock-in assay. Results from 2 independent replicates are shown. (d) Fluorescence polarization assay to detect Cas9−DNA interactions. f-DNA indicates FITC-labelled, SpCas9 PAM-containing DNA. Error bars represent mean ± SD from 3 independent replicates. For BRD0539 at 5 µM compared to DMSO, p = 0.0026 (unpaired t-test, two-tailed).

Source data

Supplementary information

Supplementary Information

Supplementary Notes and Supplementary Figs. 1–28.

Reporting Summary

Supplementary Table 1

Supplementary Tables 1–8.

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Lim, D., Zhou, Q., Cox, K.J. et al. A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Nat Cell Biol 24, 1766–1775 (2022). https://doi.org/10.1038/s41556-022-01005-8

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