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.

Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies


Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Stages of CRISPR-Cas immunity and mechanisms of Acr function.
Fig. 2: Applications of acr genes in prokaryotes.
Fig. 3: Applications and regulation of Acr proteins.
Fig. 4: Use of Acr proteins for controlling gene drives.


  1. 1.

    Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Lee, H. & Kim, J.-S. Unexpected CRISPR on-target effects. Nat. Biotechnol. 36, 703–704 (2018).

    CAS  PubMed  Google Scholar 

  5. 5.

    Ihry, R. J. et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    CAS  PubMed  Google Scholar 

  6. 6.

    Li, C. et al. HDAd5/35++ Adenovirus vector expressing anti-CRISPR peptides decreases CRISPR/Cas9 toxicity in human hematopoietic stem cells. Mol. Ther. Methods Clin. Dev. 9, 390–401 (2018). This study demonstrated that acr genes delivered into cells ex vivo can reduce Cas9-associated cytotoxicity and improve engraftment outcomes.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Chew, W. L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods 13, 868–874 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Wang, D. et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu. Rev. Virol. 4, 37–59 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

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

    CAS  PubMed  Google Scholar 

  11. 11.

    Trasanidou, D. et al. Keeping CRISPR in check: diverse mechanisms of phage-encoded anti-CRISPRs. FEMS Microbiol. Lett. 366, 1709 (2019).

    Google Scholar 

  12. 12.

    Knott, G. J. et al. Broad-spectrum enzymatic inhibition of CRISPR-Cas12a. Nat. Struct. Mol. Biol. 26, 315–321 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

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

    CAS  PubMed  Google Scholar 

  14. 14.

    Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. Nat. Rev. Microbiol. 16, 12–17 (2018).

    CAS  PubMed  Google Scholar 

  15. 15.

    Bondy-Denomy, J. et al. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins. Nature 526, 136–139 (2015). This study identified multiple mechanisms of inhibition via direct interactions with Cas proteins for the first discovered Acr proteins.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Dong, D. et al. Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein. Nature 546, 436–439 (2017). This work identified the mechanism and structure of a Cas9 inhibitor, showing AcrIIA4 binds the PAM-interacting motif of Cas9.

    CAS  PubMed  Google Scholar 

  17. 17.

    Jiang, F. et al. Temperature-responsive competitive inhibition of CRISPR-Cas9. Mol. Cell 73, 601–610.e5 (2019).

    CAS  PubMed  Google Scholar 

  18. 18.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Bondy-Denomy, J. et al. A unified resource for tracking anti-CRISPR names. CRISPR J. 1, 304–305 (2018).

    PubMed  Google Scholar 

  20. 20.

    Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158.e10 (2017). This study reported Acr proteins that inhibit SpyCas9 and demonstrated the efficacy of AcrIIA2 and AcrIIA4 in human cells.

    CAS  PubMed  Google Scholar 

  21. 21.

    Pawluk, A. et al. Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829–1838.e9 (2016). This study identified the Acr proteins that inhibit NmeCas9 and demonstrated their efficacy in human cells.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).

    CAS  PubMed  Google Scholar 

  23. 23.

    Choi, K. R. & Lee, S. Y. CRISPR technologies for bacterial systems: Current achievements and future directions. Biotechnol. Adv. 34, 1180–1209 (2016).

    CAS  PubMed  Google Scholar 

  24. 24.

    Jiang, Y. et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl. Environ. Microbiol. 81, 2506–2514 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Luo, M. L., Leenay, R. T. & Beisel, C. L. Current and future prospects for CRISPR-based tools in bacteria. Biotechnol. Bioeng. 113, 930–943 (2016).

    CAS  PubMed  Google Scholar 

  26. 26.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Luo, M. L., Mullis, A. S., Leenay, R. T. & Beisel, C. L. Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res. 43, 674–681 (2015).

    CAS  PubMed  Google Scholar 

  29. 29.

    van Belkum, A. et al. Phylogenetic distribution of CRISPR-Cas systems in antibiotic-resistant Pseudomonas aeruginosa. MBio 6, e01796–15 (2015).

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Mayo-Muñoz, D. et al. Anti-CRISPR-based and CRISPR-based genome editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10, 695 (2018). This study demonstrated the use of Acr proteins as selectable markers in viral genome engineering.

    PubMed Central  Google Scholar 

  31. 31.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Pawluk, A. et al. Disabling a type I-E CRISPR-Cas nuclease with a bacteriophage-encoded anti-CRISPR protein. MBio 8, 43 (2017).

    Google Scholar 

  33. 33.

    Louwen, R., Staals, R. H. J., Endtz, H. P., van Baarlen, P. & van der Oost, J. The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol. Mol. Biol. Rev. 78, 74–88 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Nobrega, F. L., Costa, A. R., Kluskens, L. D. & Azeredo, J. Revisiting phage therapy: new applications for old resources. Trends Microbiol. 23, 185–191 (2015).

    CAS  PubMed  Google Scholar 

  35. 35.

    Muñoz, I. V., Sarrocco, S., Malfatti, L., Baroncelli, R. & Vannacci, G. CRISPR-Cas for fungal genome editing: a new tool for the management of plant diseases. Front. Plant Sci. 10, 135 (2019).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Langner, T., Kamoun, S. & Belhaj, K. CRISPR Crops: plant genome editing toward disease resistance. Annu. Rev. Phytopathol. 56, 479–512 (2018).

    CAS  PubMed  Google Scholar 

  37. 37.

    Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Swarts, D. C. & Jinek, M. Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing. Wiley Interdiscip. Rev. RNA 9, e1481 (2018).

    Google Scholar 

  40. 40.

    Yao, R. et al. CRISPR-Cas9/Cas12a biotechnology and application in bacteria. Synth. Syst. Biotechnol. 3, 135–149 (2018).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

    CAS  PubMed  Google Scholar 

  43. 43.

    Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3, e04766 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    CAS  PubMed  Google Scholar 

  46. 46.

    Senturk, S. et al. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. Nat. Commun. 8, 14370 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  PubMed  Google Scholar 

  49. 49.

    Chen, J. S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci. Adv. 3, e1701620 (2017). This study demonstrated that AcrIIA4 can reduce off-target editing while maintaining on-target editing in human cells.

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Yang, S., Li, S. & Li, X.-J. Shortening the half-life of Cas9 maintains its gene editing ability and reduces neuronal toxicity. Cell Rep. 25, 2653–2659.e3 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).

    CAS  PubMed  Google Scholar 

  53. 53.

    Lee, J. et al. Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins. RNA rna.071704.119 (2019). This study demonstrated Cas9 inhibition with Acr proteins in mice.

  54. 54.

    Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Wu, X., Mao, S., Ying, Y., Krueger, C. J. & Chen, A. K. Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms. Genomics Proteomics Bioinformatics 17, 119–128 (2019).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

    CAS  PubMed  Google Scholar 

  60. 60.

    Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    CAS  PubMed  Google Scholar 

  61. 61.

    Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    CAS  PubMed  Google Scholar 

  62. 62.

    Li, J., Xu, Z., Chupalov, A. & Marchisio, M. A. Anti-CRISPR-based biosensors in the yeast S. cerevisiae. 1–14 (2018).

  63. 63.

    Nakamura, M. et al. Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells. Nat. Commun. 10, 194 (2019). This study demonstrates many applications of Acr proteins in eukaryotic cells, including ‘write protecting’ cells from further editing, CRISPR-based gene regulation circuits, and ligand-inducible AcrIIA4.

    PubMed  PubMed Central  Google Scholar 

  64. 64.

    Dow, L. E. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Hemphill, J., Borchardt, E. K., Brown, K., Asokan, A. & Deiters, A. Optical control of CRISPR/Cas9 gene editing. J. Am. Chem. Soc. 137, 5642–5645 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Maji, B. et al. A high-throughput platform to identify small-molecule inhibitors of CRISPR-Cas9. Cell 177, 1067–1079.e19 (2019).

    CAS  PubMed  Google Scholar 

  67. 67.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Hoffmann, M. D. et al. Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins. Nucleic Acids Res. 47, e75 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Bubeck, F. et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat. Methods 15, 924–927 (2018). This study reported an optogenetic AcrIIA4 variant that can be inactivated in cells using light.

    CAS  PubMed  Google Scholar 

  70. 70.

    Stanley, S. Y. et al. Anti-CRISPR-associated proteins are crucial repressors of anti-CRISPR transcription. Cell 178, 1452–1464.e13 (2019).

    CAS  PubMed  Google Scholar 

  71. 71.

    Hirosawa, M., Fujita, Y. & Saito, H. Cell-type-specific CRISPR activation with microRNA-responsive AcrllA4 switch. ACS Synth. Biol. 8, 1575–1582 (2019).

    CAS  PubMed  Google Scholar 

  72. 72.

    Burt, A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc. Biol. Sci. 270, 921–928 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Gantz, V. M. et al. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc. Natl Acad. Sci. USA 112, E6736–E6743 (2015).

    CAS  PubMed  Google Scholar 

  74. 74.

    Hammond, A. et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat. Biotechnol. 34, 78–83 (2016).

    CAS  PubMed  Google Scholar 

  75. 75.

    Esvelt, K. M., Smidler, A. L., Catteruccia, F. & Church, G. M. Concerning RNA-guided gene drives for the alteration of wild populations. Elife 3, 20131071 (2014).

    Google Scholar 

  76. 76.

    Akbari, O. S. et al. BIOSAFETY. Safeguarding gene drive experiments in the laboratory. Science 349, 927–929 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Basgall, E. M. et al. Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae. Microbiology 164, 464–474 (2018). This study demonstrated the ability of AcrIIA2 and AcrIIA4 to halt gene drives in yeast.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Johnston, R. K. et al. Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection. Biosens. Bioelectron. 141, 111361 (2019).

    CAS  PubMed  Google Scholar 

  79. 79.

    Palmer, D. J., Turner, D. L. & Ng, P. Production of CRISPR/Cas9-mediated self-cleaving helper-dependent adenoviruses. Mol. Ther. Methods Clin. Dev. 13, 432–439 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Smargon, A. A. et al. Cas13b Is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol. Cell 65, 618–630.e7 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Pawluk, A. et al. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species. Nat. Microbiol. 1, 16085 (2016).

    CAS  PubMed  Google Scholar 

  82. 82.

    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). This study identified phage proteins with Acr function.

    CAS  PubMed  Google Scholar 

  83. 83.

    Guo, T. W. et al. Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Cell 171, 414–426.e12 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. 84.

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

    CAS  PubMed  Google Scholar 

  85. 85.

    Pawluk, A., Bondy-Denomy, J., Cheung, V. H. W., Maxwell, K. L. & Davidson, A. R. A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. MBio 5, e00896 (2014). e00896–e14.

    PubMed  PubMed Central  Google Scholar 

  86. 86.

    Fuchsbauer, O. et al. Cas9 Allosteric inhibition by the anti-CRISPR protein AcrIIA6. Mol. Cell 76, 922–937.e7 (2019).

    CAS  PubMed  Google Scholar 

  87. 87.

    Hynes, A. P. et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat. Commun. 9, 2919 (2018).

    PubMed  PubMed Central  Google Scholar 

  88. 88.

    Lee, J. et al. Potent Cas9 inhibition in bacterial and human cells by AcrIIC4 and AcrIIC5 anti-CRISPR proteins. MBio 9, 1239 (2018).

    Google Scholar 

  89. 89.

    Sun, W. et al. Structures of Neisseria meningitidis Cas9 complexes in catalytically poised and anti-CRISPR-inhibited states. Mol. Cell 76, 938–952.e5 (2019).

    CAS  PubMed  Google Scholar 

  90. 90.

    Thavalingam, A. et al. Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2. Nat. Commun. 10, 2806–2811 (2019).

    PubMed  PubMed Central  Google Scholar 

  91. 91.

    Zhu, Y. et al. Diverse mechanisms of CRISPR-Cas9 inhibition by type IIC anti-CRISPR proteins. Mol. Cell 74, 296–309.e7 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Forsberg, K. J. et al. Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome. Elife 8, 1709 (2019).

    Google Scholar 

  93. 93.

    Ka, D., An, S. Y., Suh, J.-Y. & Bae, E. Crystal structure of an anti-CRISPR protein, AcrIIA1. Nucleic Acids Res. 46, 485–492 (2018).

    CAS  PubMed  Google Scholar 

  94. 94.

    Hynes, A. P. et al. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9. Nat. Microbiol. 2, 1374–1380 (2017).

    CAS  PubMed  Google Scholar 

  95. 95.

    Uribe, R. V. et al. Discovery and characterization of Cas9 inhibitors disseminated across seven bacterial phyla. Cell Host Microbe 25, 233–241.e5 (2019).

    CAS  PubMed  Google Scholar 

  96. 96.

    Bhoobalan-Chitty, Y., Johansen, T. B., Di Cianni, N. & Peng, X. Inhibition of type III CRISPR-Cas immunity by an archaeal virus-encoded anti-CRISPR protein. Cell 179, 448–458.e11 (2019).

    CAS  PubMed  Google Scholar 

  97. 97.

    Zhang, H. et al. Structural basis for the inhibition of CRISPR-Cas12a by anti-CRISPR proteins. Cell Host Microbe 25, 815–826.e4 (2019).

    CAS  PubMed  Google Scholar 

  98. 98.

    Watters, K. E., Fellmann, C., Bai, H. B., Ren, S. M. & Doudna, J. A. Systematic discovery of natural CRISPR-Cas12a inhibitors. Science 9, eaau5138 (2018).

    Google Scholar 

  99. 99.

    Wandera, K. G. et al. An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system. Methods (2019).

Download references


We thank M. Pinilla-Redondo, who made the figures for this manuscript. Acr research in the Bondy-Denomy lab was supported by the University of California San Francisco Program for Breakthrough in Biomedical Research, funded in part by the Sandler Foundation, by an NIH Office of the Director Early Independence Award DP5-OD021344, by NIH R01GM127489, and by DARPA HR0011-17-2-0043. N.D.M. was supported by NIH F32GM133127, B.C. was supported by the Eötvös National Scholarship of Hungary and a Marie Skłodowska-Curie Actions Individual Global Fellowship (number 844093) of the Horizon 2020 Research Program of the European Commission. R.P.R. was funded by Joint Programming Initiative-Antimicrobial Resistance (JIP-AMR; DARWIN project, #7044-00004B), the Innovation Fund Denmark (Trojan Horse Project, #5157-00005B).

Author information




N.D.M. wrote the sections on the applications of Acr proteins for eukaryotic and in vitro systems and regulation of Acr proteins, and the informational boxes on the advantages and limitations of Acr proteins. R.P.R. wrote the introduction, informational box for Acr protein discovery, and Table 1. B.C. wrote the sections on controlling gene drives with Acr proteins and applications of Acr proteins for prokaryotic systems. J.B.-D. supervised and wrote the manuscript with N.D.M, R.P.R. and B.C. All authors contributed to figure content and edited the manuscript.

Corresponding author

Correspondence to Joseph Bondy-Denomy.

Ethics declarations

Competing interests

J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics and a scientific advisory board member and co-founder of Acrigen Biosciences. J.B.-D. and N.D.M. have filed patents on technology related to anti-CRISPR proteins. R.P.R. is a consultant for Ancilia Inc.

Additional information

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Marino, N.D., Pinilla-Redondo, R., Csörgő, B. et al. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nat Methods 17, 471–479 (2020).

Download citation

Further reading


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