Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An anti-CRISPR protein disables type V Cas12a by acetylation


Phages use anti-CRISPR proteins to deactivate the CRISPR–Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR–Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR–Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR–Cas inhibition and suggests an evolutionary arms race between phages and bacteria.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: AcrVA5 inhibits MbCas12a activity through acetyltransferase activity.
Fig. 2: AcrVA5 adopts a typical acetyltransferase structure.
Fig. 3: Lys635 acetylation by AcrVA5 sterically blocks MbCas12a from PAM recognition.
Fig. 4: Mutation of the critical PAM-recognition lysine residue to arginine results in escape of MbCas12a inhibition by AcrVA5.

Data availability

The atomic coordinates and structure factors of AcrVA5 have been deposited in the Protein Data Bank under accession code 6IUF. The atomic coordinates of acetylated MbCas12a have been deposited in the Protein Data Bank under accession code 6IV6. The corresponding maps have been deposited in the Electron Microscopy Data Bank under accession code EMD-9742. The data sets generated and analyzed are available from the corresponding authors on request.


  1. Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  Google Scholar 

  2. Marraffini, L. A. CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61 (2015).

    Article  CAS  Google Scholar 

  3. Westra, E. R. et al. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012).

    Article  CAS  Google Scholar 

  4. Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82, 237–266 (2013).

    Article  CAS  Google Scholar 

  5. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  6. Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    Article  CAS  Google Scholar 

  7. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  Google Scholar 

  8. Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).

    Article  CAS  Google Scholar 

  9. Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016).

    Article  CAS  Google Scholar 

  10. Tang, X. et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17103 (2017).

    Article  Google Scholar 

  11. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  Google Scholar 

  12. Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).

    Article  CAS  Google Scholar 

  13. Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

    Article  CAS  Google Scholar 

  14. He, F. et al. Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity. Nat Microbiol 3, 461–469 (2018).

    Article  CAS  Google Scholar 

  15. Pawluk, A. et al. Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829–1838 e9 (2016).

    Article  CAS  Google Scholar 

  16. Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158 e10 (2017).

    Article  CAS  Google Scholar 

  17. Dong et al. Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature 546, 436–439 (2017).

    Article  CAS  Google Scholar 

  18. Stanley, S. Y. & Maxwell, K. L. Phage-encoded anti-CRISPR defenses. Annu. Rev. Genet. 52, 445–464 (2018).

    Article  CAS  Google Scholar 

  19. Gunaratne, R. et al. Patient dissatisfaction following total knee arthroplasty: a systematic review of the literature. J. Arthroplasty 32, 3854–3860 (2017).

    Article  Google Scholar 

  20. Watters, K. E., Fellmann, C., Bai, H. B., Ren, S. M. & Doudna, J. A. Systematic discovery of natural CRISPR-Cas12a inhibitors. Science 362, 236–239 (2018).

    Article  CAS  Google Scholar 

  21. Marino, N. D. et al. Discovery of widespread type I and type V CRISPR-Cas inhibitors. Science 362, 240–242 (2018).

    Article  CAS  Google Scholar 

  22. Shmakov, S. A. et al. The CRISPR spacer space is dominated by sequences from species-specific mobilomes. MBio 8, e01397-17 (2017).

    Article  Google Scholar 

  23. Heussler, G. E. & O’Toole, G. A. Friendly fire: biological functions and consequences of chromosomal targeting by CRISPR-cas systems. J. Bacteriol. 198, 1481–1486 (2016).

    Article  CAS  Google Scholar 

  24. Zhang, F. et al. CRISPRminer is a knowledge base for exploring CRISPR-Cas systems in microbe and phage interactions. Commun. Biol. 1, 180 (2018).

    Article  Google Scholar 

  25. Yang, H. & Patel, D. J. Inhibition mechanism of an anti-CRISPR suppressor AcrIIA4 targeting SpyCas9. Mol. Cell 67, 117–127 e5 (2017).

    Article  CAS  Google Scholar 

  26. Salah Ud-Din, A. I., Tikhomirova, A. & Roujeinikova, A. Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). Int. J. Mol. Sci. 17, E1018 (2016).

  27. Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44, W351–W355 (2016).

    Article  CAS  Google Scholar 

  28. Magin, R. S., Liszczak, G. P. & Marmorstein, R. The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD. Structure 23, 332–341 (2015).

    Article  CAS  Google Scholar 

  29. Yamano, T. et al. Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1. Mol. Cell 67, 633–645 e3 (2017).

    Article  CAS  Google Scholar 

  30. Chowdhury, S. et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell 169, 47–57 e11 (2017).

    Article  CAS  Google Scholar 

  31. Harrington, L. B. et al. A broad-spectrum inhibitor of CRISPR-Cas9. Cell 170, 1224–1233.e15 (2017).

    Article  CAS  Google Scholar 

  32. Maxwell, K. L. et al. The solution structure of an anti-CRISPR protein. Nat .Commun. 7, 13134 (2016).

    Article  CAS  Google Scholar 

  33. Bondy-Denomy, J. et al. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins. Nature 526, 136–139 (2015).

    Article  CAS  Google Scholar 

  34. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  Google Scholar 

  35. Xiong, B. et al. Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol. Biofuels 11, 172 (2018).

    Article  Google Scholar 

  36. Feng, X., Zhao, D., Zhang, X., Ding, X. & Bi, C. CRISPR/Cas9 assisted multiplex genome editing technique in Escherichia coli. Biotechnol. J. 13, e1700604 (2018).

    Article  Google Scholar 

  37. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  38. McCoy, A. J. et al. Phaser crystallographic software. J .Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  40. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).

    Article  Google Scholar 

  41. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  42. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  43. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  Google Scholar 

  44. Shaikh, T. R. et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat. Protoc. 3, 1941–1974 (2008).

    Article  CAS  Google Scholar 

  45. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

  46. Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. eL ife 7, e35383 (2018).

  47. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  Google Scholar 

  48. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  49. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  50. Hovmoller, S., Zhou, T. & Ohlson, T. Conformations of amino acids in proteins. Acta Crystallogr. D 58, 768–776 (2002).

    Article  Google Scholar 

  51. Couvin, D. et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 46, W246–W251 (2018).

    Article  Google Scholar 

  52. Edgar, R. C. PILER-CR: fast and accurate identification of CRISPR repeats. BMC Bioinformatics 8, 18 (2007).

    Article  Google Scholar 

  53. Arndt, D. et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res. 44, W16–W21 (2016).

    Article  CAS  Google Scholar 

Download references


We thank J. He at the Shanghai Synchrotron Radiation Facility (SSRF) for help with data collection. We thank the Core Facilities at the School of Life Sciences, Peking University, for assistance with negative-staining EM and the Electron Microscopy Laboratory and the cryo-EM platform of Peking University for help with cryo-EM data collection. The computation was supported by the High-performance Computing Platform of Peking University. We thank X. Meng and H. Deng at the Center of Biomedical Analysis, Tsinghua University, for protein MS analysis. We thank J. Chai for critical reading of the manuscript. This research was funded by the National Natural Science Foundation of China grant no. 31825008 and 31422014 to Z.H.; 31700655 to N.L. N.L. is supported by Young Elite Scientists Sponsorship Program by CAST.

Author information

Authors and Affiliations



F.Z. and F.Z. conducted bioinformatics analysis. L.D. and X.G. expressed, purified and characterized proteins, and crystallized AcrVA5. Y.Z. and N.L. carried out crystallographic and cryo-EM studies. R.K. and L.Y. performed in vivo experiments. L.D. and X.G. performed biochemical assays. L.D., X.G., Z.H., N.G. and Z.H. wrote the paper. All authors contributed to the manuscript preparation. Z.H. designed the experiments.

Corresponding author

Correspondence to Zhiwei Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Bioinformatics approach for discovering anti-CRISPR candidates for the V-A CRISPR-Cas12a system (AcrVAs) and identification of anti-CRISPR genes.

a, Statistics of the type of genomic elements that anti-CRISPRs have been discovered to inhibit type I or II CRISPR-Cas system (based on the anti-CRISPRs collection reported in b, CRISPRminer pipeline was used to predict the V-A CRISPR-Cas systems (including CRISPR arrays, Cas locus and their (sub)types), find spacers with identifiable proto-spacers target their own prophage regions (self-targeting, ST) or prophage regions on other genomes (non-self-targeting, NST). c, In vitro MbCas12a inhibition assay was used to identify the anti-CRISPRs from 49 purified proteins (49 candidates). MbCas12a was first incubated with anti-CRISPR candidates at 1:8 molar ratio. Then crRNA and dsDNA substrate were added and incubated. The reactions were stopped by adding loading buffer for denaturing gel and the reaction mixtures were run on TBE-Urea polyacrylamide gels and visualized by EB staining. Data shown are representative of three independent experiments. d, Plasmid-targeting assay with 49 gene products (candidates 1-50, except 38) from M. bovoculi 22581 prophage2 in E. coli. Survival (CFU: colony-forming unit) of bacteria was calculated in the presence of candidate and targeted plasmids. Non-targeted plasmid co-transformed with candidate 49 (NT+49), targeted plasmid co-transformed with AcrIIA4 (A4), and only targeted plasmid (-) were controls of the assay. The inhibitory effects of AcrIIA4 and candidate 50 against SpCas9 were also tested by four groups: non-targeted plasmid co-transformed with AcrIIA4 (NT+A4), targeted plasmid co-transformed with AcrIIA4 (A4), targeted plasmid co-transformed with candidate 50 (50) and only targeted plasmid (-). Error bars represent the mean±SD of three biological replicates.

Supplementary Figure 2 Inhibition of MbCas12a by AcrVA4 and AcrVA5.

a, Inhibition assays for MbCas12a by AcrVA4 and AcrVA5. Left panel: crRNA was added to buffer containing MbCas12a following addition of AcrVA4 or AcrVA5. Right panel: MbCas12a was pre-incubated with crRNA before AcrVA4 or AcrVA5 was added. After reaction, the reaction mixtures were loaded onto 8% denature polyacrylamide gels. Molar ratios of an anti-CRISPR protein and MbCas12a are shown at the top of each lane. b, Electrophoretic mobility shift assay (EMSA) was used to test the binding of MbCas12a with crRNA or dsDNA in the presence of anti-CRISPR protein AcrVA4. MbCas12a or MbCas12a (D874A/E968A)-crRNA complex was first incubated with increasing amounts of AcrVA4, then crRNA (upper panel) or fluorophore-labelled dsDNA (middle panel) was added and incubated. Nucleic acid-bound MbCas12a complexes were resolved from free nucleic acid by native gel electrophoresis. CrRNA was visualized by ethidium bromide staining (upper panel), and dsDNA was visualized by a fluorescence imaging machine (600 nm) (middle panel). Coomassie blue staining of AcrVA4 at different molar ratios was shown in lower panel. Molar ratios of MbCas12a:AcrVA4 are shown at the top of each lane. Data shown is representative of three independent experiments.

Supplementary Figure 3 The structures of AcrVA5 and the PAM-interacting domain from LbCas12a.

a, Shown is the result of gel filtration experiment after the reaction of AcrVA5 and crRNA-bound MbCas12a. Top, UV absorbance (280 nm) peaks from size-exclusion chromatography. Bottom, Coomassie blue staining following SDS–PAGE of the peak fractions from gel filtration shown on the top. b, The overall structure of the acetyl-CoA-bound AcrVA5 complex shown in cartoon, and two acetyl-CoA molecules are shown as sticks. The two AcrVA5 molecules in the structure were colored cyan (left) and green (right), respectively. c, Detailed interactions of the PAM base pairs with Lys607. Hydrogen bonds are shown as dashed lines. Recognition of the 5’-TTTA-3’ PAM by LbCas12a (PDB: 5XUS).

Supplementary Figure 4 Workflow of the data processing of the AcrVA5-acetylated MbCas12a in complex with crRNA.

a, A representative cryo-EM image of MbCas12a-crRNA complex. b, Representative class averages of 2D classification of auto-picked particles. Particles included for further processing were indicated by red boxes. c, Workflow of the image processing, including unsupervised and supervised 3D classification, 3D refinement and CTF refinement. See more details in Methods.

Supplementary Figure 5 AcrVA5 inhibits the Cas12a strain with lysine but not arginine for recognition of PAM.

a, Time course of cleavage inhibition assays were used to test the inhibition activities of AcrVA5 against WT and R608K mutant Lb2Cas12a. The reactions were stopped by adding loading buffer for denaturing gel and the reaction mixtures were run on TBE-urea polyacrylamide gels and visualized by ethidium bromide staining. Lb2Cas12a from strain Lachnospiraceae bacterium COE1 denotes a close LbCas12a ortholog (45% identity). b, Time course of cleavage inhibition assays were used to test the inhibition activities of AcrVA5 against WT and K595R mutant LbCas12a. The reactions were stopped by adding loading buffer for denaturing gel and the reaction mixtures were run on TBE-urea polyacrylamide gels and visualized by ethidium bromide staining.

Supplementary information

Supplementary Figures, Supplementary Tables, Supplementary Note and Supplementary Dataset

Supplementary Figures 1–5, Supplementary Tables 1–2, Supplementary Note 1 and Supplementary Dataset 1

Reporting Summary

Supplementary Dataset 2

MS data of AcrVA5-modified MbCas12a

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dong, L., Guan, X., Li, N. et al. An anti-CRISPR protein disables type V Cas12a by acetylation. Nat Struct Mol Biol 26, 308–314 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing