Recent advances in noncanonical inhibition mechanisms of anti-CRISPR proteins

Abstract

The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage–host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.

Keywords

anti-CRISPR / CRISPR-Cas / noncanonical inhibition / phage–host arms race / structural mechanism

References

[1] Murtazalieva K, Mu A, Petrovskaya A, Finn RD. The growing repertoire of phage anti-defence systems. Trends Microbiol. 2024; 32: 1212–1228.

[2] Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008; 321: 960–964.

[3] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337: 816–821.

[4] Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature. 2013; 493: 429–432.

[5] Kempton HR, Qi LS. When genome editing goes off-target. Science. 2019; 364: 234–236.

[6] Li C, Psatha N, Gil S, Wang H, Papayannopoulou T, Lieber A, et al. HDAd5/35⁺⁺ adenovirus vector expressing anti-CRISPR peptides decreases CRISPR/Cas9 toxicity in human hematopoietic stem cells. Mol Ther Methods Clin Dev. 2018; 9: 390–401.

[7] Bondy-Denomy J, Davidson AR, Doudna JA, Fineran PC, Maxwell KL, Moineau S, et al. A unified resource for tracking anti-CRISPR names. CRISPR J. 2018; 1: 304–305.

[8] Yang L, Zhang Y, Yin P, Feng Y. Structural insights into the inactivation of the type I-F CRISPR-Cas system by anti-CRISPR proteins. RNA Biol. 2021; 18: 562–573.

[9] Yin P, Zhang Y, Yang L, Feng Y. Non-canonical inhibition strategies and structural basis of anti-CRISPR proteins targeting type I CRISPR-Cas systems. J Mol Biol. 2023; 435: 167996.

[10] Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987; 169: 5429–5433.

[11] Jansen R, Embden JDA van, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002; 43: 1565–1575.

[12] Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315: 1709–1712.

[13] Mojica FJM, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000; 36: 244–246.

[14] Nussenzweig PM, Marraffini LA. Molecular mechanisms of CRISPR-Cas immunity in bacteria. Annu Rev Genet. 2020; 54: 93–120.

[15] Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct. 2011; 6: 38.

[16] Makarova KS, Shmakov SA, Wolf YI, Mutz P, Altae-Tran H, Beisel CL, et al. An updated evolutionary classification of CRISPR–Cas systems including rare variants. Nat Microbiol. 2025; 10: 3346–3361.

[17] Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471: 602–607.

[18] Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011; 39: 9275–9282.

[19] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819–823.

[20] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339: 823–826.

[21] Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018; 19: 770–788.

[22] Dong C, Hao G-F, Hua H-L, Liu S, Labena AA, Chai G, et al. Anti-CRISPRdb: a comprehensive online resource for anti-CRISPR proteins. Nucleic Acids Res. 2018; 46: D393–D398.

[23] Jia N, Patel DJ. Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Nat Rev Mol Cell Biol. 2021; 22: 563–579.

[24] Bondy-Denomy J, Garcia B, Strum S, Du M, Rollins MF, Hidalgo-Reyes Y, et al. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins. Nature. 2015; 526: 136–139.

[25] Chowdhury S, Carter J, Rollins MF, Golden SM, Jackson RN, Hoffmann C, et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell. 2017; 169: 47–57.

[26] Guo TW, Bartesaghi A, Yang H, Falconieri V, Rao P, Merk A, et al. Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Cell. 2017; 171: 414–426.e12.

[27] Wang X, Yao D, Xu JG, Li AR, Xu J, Fu P, et al. Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3. Nat Struct Mol Biol. 2016; 23: 868–870.

[28] Cao X, Gao Z, Yin P, Wang H, Yang L. Crystal structure and inhibition mechanism of AcrIIA11. Biochem Biophys Res Commun. 2025; 772: 152073.

[29] Deng X, Sun W, Li X, Wang J, Cheng Z, Sheng G, et al. An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding. Nat Commun. 2024; 15: 1806.

[30] Kim GE, Park HH. AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain. Nucleic Acids Res. 2024; 52: 6459–6471.

[31] Sun W, Cheng Z, Wang J, Yang J, Li X, Wang J, et al. AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation. Proc Natl Acad Sci USA. 2023; 120:e2303675120.

[32] Li X, Liao F, Gao J, Song G, Zhang C, Ji N, et al. Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4. Nucleic Acids Res. 2023; 51: 9442–9451.

[33] Chou-Zheng L, Howell O, Boyle TA, Hossain M, Walker FC, Sheriff EK, et al. AcrIIIA1 is a protein-RNA anti-CRISPR complex that targets core Cas and accessory nucleases. Nucleic Acids Res. 2024; 52: 13490–13514.

[34] Liu J, Li Q, Wang X, Liu Z, Ye Q, Liu T, et al. An archaeal virus-encoded anti-CRISPR protein inhibits type III-B immunity by inhibiting Cas RNP complex turnover. Nucleic Acids Res. 2023; 51: 11783–11796.

[35] Wandera KG, Schmelz S, Migur A, Kibe A, Lukat P, Achmedov T, et al. AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack. Mol Cell. 2025; 85: 1162–1175.

[36] Allemailem K, Almatroudi A, Alrumaihi F, Alradhi A, Theyab A, Algahtani M, et al. Current updates of CRISPR/Cas system and anti-CRISPR proteins: innovative applications to improve the genome editing strategies. Int J Nanomed. 2024; 19: 10185–10212.

[37] Niu Y, Yang L, Gao T, Dong C, Zhang B, Yin P, et al. A type I-F anti-CRISPR protein inhibits the CRISPR-Cas surveillance complex by ADP-ribosylation. Mol Cell. 2020; 80: 512–524.

[38] Dong L, Guan X, Li N, Zhang F, Zhu Y, Ren K, et al. An anti-CRISPR protein disables type V Cas12a by acetylation. Nat Struct Mol Biol. 2019; 26: 308–314.

[39] Yang L, Zhang L, Yin P, Ding H, Xiao Y, Zeng J, et al. Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24. Nat Commun. 2022; 13: 1931.

[40] Xie Y, Zhang L, Gao Z, Yin P, Wang H, Li H, et al. AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition. Nat Chem Biol. 2022; 18: 670–677.

[41] Osuna BA, Karambelkar S, Mahendra C, Christie KA, Garcia B, Davidson AR, et al. Listeria phages induce Cas9 degradation to protect lysogenic genomes. Cell Host Microbe. 2020; 28: 31–40.

[42] Forsberg KJ, Schmidtke DT, Werther R, Uribe RV, Hausman D, Sommer MOA, et al. The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity. PLoS Biol. 2021; 19:e3001428.

[43] Zhang H, Li Z, Daczkowski CM, Gabel C, Mesecar AD, Chang L. Structural basis for the inhibition of CRISPR-Cas12a by anti-CRISPR proteins. Cell Host Microbe. 2019; 25: 815–826.e4.

[44] Bi M, Su W, Li J, Mo X. Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5. Nat Commun. 2024; 15: 3256.

[45] Philippe C, Morency C, Plante P-L, Zufferey E, Achigar R, Tremblay DM, et al. A truncated anti-CRISPR protein prevents spacer acquisition but not interference. Nat Commun. 2022; 13: 2802.

[46] Drabavicius G, Sinkunas T, Silanskas A, Gasiunas G, Venclovas Č, Siksnys V. DnaQ exonuclease-like domain of Cas2 promotes spacer integration in a type I-E CRISPR-Cas system. EMBO Rep. 2018; 19:e45543.

[47] Dyda F, Hickman AB. Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA. Mob DNA. 2015; 6: 9.

[48] Wang J, Li J, Zhao H, Sheng G, Wang M, Yin M, et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR-Cas systems. Cell. 2015; 163: 840–853.

[49] Hynes AP, Rousseau GM, Agudelo D, Goulet A, Amigues B, Loehr J, et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat Commun. 2018; 9: 2919.

[50] Fuchsbauer O, Swuec P, Zimberger C, Amigues B, Levesque S, Agudelo D, et al. Cas9 allosteric inhibition by the anti-CRISPR protein AcrIIA6. Mol Cell. 2019; 76: 922–937.

[51] Marino ND, Talaie A, Carion H, Johnson MC, Zhang Y, Silas S, et al. Translation-dependent downregulation of Cas12a mRNA by an anti-CRISPR protein. BioRxiv. 2024. https://doi.org/10.1101/2022.11.29.518452.

[52] Chen P, Sun W, Cheng Z, Yang J, Wang M, Wang J-Y, et al. Structural study on anti-CRISPR protein AcrVA2. Prog Biochem Biophys. 2021; 48: 77–87.

[53] Leroy M, Piton J, Gilet L, Pellegrini O, Proux C, Coppée J-Y, et al. Rae1/YacP, a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis. EMBO J. 2017; 36: 1167–1181.

[54] Trost CN, Yang J, Garcia B, Hidalgo-Reyes Y, Fung BCM, Wang J, et al. An anti-CRISPR that pulls apart a CRISPR–Cas complex. Nature. 2024; 632: 375–382.

[55] Zhang K, Wang S, Li S, Zhu Y, Pintilie GD, Mou T-C, et al. Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM. Proc Natl Acad Sci USA. 2020; 117: 7176–7182.

[56] Liu X, Zhang L, Xiu Y, Gao T, Huang L, Xie Y, et al. Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR-Cas surveillance complex. Nucleic Acids Res. 2021; 49: 10178–10191.

[57] Gabel C, Li Z, Zhang H, Chang L. Structural basis for inhibition of the type I-F CRISPR-Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14. Nucleic Acids Res. 2021; 49: 584–594.

[58] Kim GE, Lee SY, Birkholz N, Kamata K, Jeong J-H, Kim Y-G, et al. Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24. Nucleic Acids Res. 2022; 50: 11344–11358.

[59] Mukherjee IA, Gabel C, Noinaj N, Bondy-Denomy J, Chang L. Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor. Nat Chem Biol. 2022; 18: 1417–1424.

[60] Jiang C, Yu C, Sun S, Lin J, Cai M, Wei Z, et al. A new anti-CRISPR gene promotes the spread of drug-resistance plasmids in Klebsiella pneumoniae. Nucleic Acids Res. 2024; 52: 8370–8384.

[61] Katz MA, Sawyer EM, Oriolt L, Kozlova A, Williams MC, Margolis SR, et al. Diverse viral cas genes antagonize CRISPR immunity. Nature. 2024; 634: 677–683.

[62] Chen DF, Roe LT, Li Y, Borges AL, Zhang JY, Babbar P, et al. AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid. mBio. 2025; 16:e0169825.

[63] Wang X, Li X, Ma Y, He J, Liu X, Yu G, et al. Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18. Nucleic Acids Res. 2022; 50: 512–521.

[64] Athukoralage JS, McMahon SA, Zhang C, Grüschow S, Graham S, Krupovic M, et al. An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature. 2020; 577: 572–575.

[65] Kang X, Yin L, Zhuang S, Hu T, Wu Z, Zhao G, et al. Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation. Cell Discov. 2022; 8: 45.

[66] Kim DY, Lee SY, Ha HJ, Park HH. AcrIE7 inhibits the CRISPR-Cas system by directly binding to the R-loop single-stranded DNA. Proc Natl Acad Sci USA. 2025; 122:e2423205122.

[67] McGinn J, Marraffini LA. CRISPR-Cas systems optimize their immune response by specifying the site of spacer integration. Mol Cell. 2016; 64: 616–623.

[68] Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat Struct Mol Biol. 2014; 21: 528–534.

[69] He F, Bhoobalan-Chitty Y, Van LB, Kjeldsen AL, Dedola M, Makarova KS, et al. Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity. Nat Microbiol. 2018; 3: 461–469.

[70] Manav MC, Van LB, Lin J, Fuglsang A, Peng X, Brodersen DE. Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein. Nat Commun. 2020; 11: 5993.

[71] Workman RE, Stoltzfus MJ, Keith NC, Euler CW, Bondy-Denomy J, Modell JW. Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense. Cell Rep. 2024; 43:113849.

[72] Liu C, Wang R, Li J, Cheng F, Shu X, Zhao H, et al. Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins. Cell Host Microbe. 2023; 31: 1481–1493.

[73] Li M, Gong L, Cheng F, Yu H, Zhao D, Wang R, et al. Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems. Science. 2021; 372:eabe5601.

[74] Shu X, Wang R, Li Z, Xue Q, Wang J, Liu J, et al. CRISPR-repressed toxin–antitoxin provides herd immunity against anti-CRISPR elements. Nat Chem Biol. 2025; 21: 337–347.

[75] Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024; 630: 493–500.

[76] Sahakyan H, Makarova KS, Koonin EV. Search for origins of anti-CRISPR proteins by structure comparison. CRISPR J. 2023; 6: 222–231.

[77] Johnson MC, Hille LT, Kleinstiver BP, Meeske AJ, Bondy-Denomy J. Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages. Mol Cell. 2022; 82: 2161–2166.

[78] Martínez-Alvarez L, Stickel D, Salegi-Díez A, Bhoobalan-Chitty Y, Shen WZ, Peng X. Natively expressed AcrIII-1 does not function as an anti-CRISPR protein. Nature. 2025; 640: E8–E14.

[79] Li D, Xiao Y, Fedorova I, Xiong W, Wang Y, Liu X, et al. Single phage proteins sequester signals from TIR and cGAS-like enzymes. Nature. 2024; 635: 719–727.

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