Abstract
Bacteria encode diverse defence systems, including CRISPR–Cas, to recognize and cleave the DNA of bacteriophages (phages) and other mobile genetic elements1. In response, phages encode anti-CRISPR (Acr) proteins that inhibit CRISPR–Cas activity by blocking DNA binding or cleavage2. Here we report an unexpected mechanism by which the anti-CRISPR AcrVA2 inhibits Cas12a biogenesis. AcrVA2 binds conserved and functionally important amino acid residues near the Cas12a N-terminus and triggers selective degradation of cas12a mRNA as it is translated. Additionally, conserved residues in the AcrVA2 C-terminal domain enable co-sedimentation with ribosomes and polysomes, which is required to achieve targeted co-translational mRNA degradation. The AcrVA2 C-terminal domain is broadly conserved in homologs encoded by diverse mobile genetic elements, typically in hosts that lack cas12a, suggesting that these homologues may recognize and downregulate alternative substrates in other bacteria. These findings reveal a novel mechanism for molecular conflict and gene regulation in bacteria.
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Data availability
Transcriptomic data have been deposited in the NCBI Sequence Read Archive under BioProject accession PRJNA1001058. The proteins used to construct the phylogenetic tree (https://doi.org/10.6084/m9.figshare.31680328) and the mass spectrometry data (https://doi.org/10.6084/m9.figshare.31680478) are publicly available on Figshare41,42. Uncropped images of gels and blots are presented in Supplementary Fig. 1. Source data are provided for Figs. 1c,d and 2i,l and Extended Data Figs. 1c, 2a–d, 4a,b,5a,b and 9c,d. Amino acid alignments of AcrVA2 homologues are publicly available on Figshare (https://doi.org/10.6084/m9.figshare.31684630)43. Sequences of AcrVA2 homologues used to generate these amino acid alignments are listed in Supplementary Table 2. Nucleotide alignments of different cas12a variants downregulated by AcrVA2 are publicly available on Figshare (https://doi.org/10.6084/m9.figshare.31684813)44. Source data are provided with this paper.
Code availability
This paper does not report any original code.
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Acknowledgements
We thank members of the Bondy-Denomy laboratory and C. Gross laboratory for thoughtful discussions; K. Watters for providing plasmids; J. Peters and A. Buskirk for reagents and technical advice; M. Paredes and D. Le for preparing the media used in this work; and K. Lynch for providing us with the necessary facilities and equipment to perform radioactivity experiments. Sequencing was performed at the UCSF CAT. This work was supported by the US National Institutes of Health (R01GM127489), the Vallee Foundation and the Searle Scholarship, and the Defense Advanced Research Projects Agency (DARPA) award HR0011-17-2-0043. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the DARPA Safe Genes program (HR0011-17-2-0043). N.D.M. was supported by the National Institutes of Health awards F32GM133127, K99GM143476 and R00GM143476. M.G. was funded by the Federal Ministry of Research, Technology and Space of Germany under the Microbial Stargazing project (ref. 01KX2324). J.V. was supported by the Gottfried Wilhelm Leibniz Award (DFG Vo875‐18) and SPP2330 (DFG Vo875‐23/2). J.V. is a member of the DFG Cluster of Excellence NUCLEATE for Nucleic Acid Sciences and Technologies (Project-ID 533767322 – EXC 3113/1).
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N.D.M. and J.B.-D. conceptualized the project. N.D.M., M.G., A.D.S., A.F.T., K.Z., S.S., M.C.J. and D.L.S. contributed to the experimental design. N.D.M. and J.B.-D. acquired funding. N.D.M. performed the plasmid construction, strain engineering, phage plaque assays, western blots, immunoprecipitations, qRT–PCR, northern blots, RNA sequencing library preparation, growth assays, and structural and sequence alignments. A.T. performed the plasmid construction, strain engineering, in vitro cleavage assays, qRT–PCR, western blots and plaque assays. M.G. and L.B. performed the sucrose gradients and western blots. J.L.R. performed the plasmid construction, strain engineering, northern blots, western blots and growth assays. A.D.S. performed the plasmid construction, strain engineering, western blots and qRT–PCR. A.F.T. and K.Z. performed the northern and western blots. H.C. performed the immunoprecipitations and western blots. T.J.A. performed the plaque assays, western blots and qRT–PCR. J.L. performed the strain engineering, plaque assays, western blots and nucleotide alignments. S.H. performed the strain engineering and plaque assays. M.C.J. generated the phylogenetic tree. S.S. performed the RNA sequencing analysis. L.Y. plotted the transcriptomics data. K.-H.C. performed the liquid chromatography–mass spectrometry and analysis. Y.Z. performed the protein purification and in vitro assays. N.D.M., J.B.-D., J.V., M.G. and D.L.S. oversaw the different aspects of the project. N.D.M. and J.B.-D. wrote the manuscript with input from all authors.
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The UCSF has filed a patent on the use of inhibitors of CRISPR–Cas12a, on which N.D.M. and J.B.-D. are listed as inventors. J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a consultant to LeapFrog Bio, and a scientific advisory board member and co-founder of Acrigen Biosciences. The Bondy-Denomy laboratory received research support from Felix Biotechnology. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 AcrVA2 inhibits and downregulates diverse variants of cas12a.
(a) Phage plaque assay using ten-fold serial dilution of phage to assess Cas12a inhibition by AcrVA2 (or homolog AcrVA2.1) compared to controls. 33362, 237, and 58069 refer to the Moraxella bovoculi strains that encode the corresponding Cas12a homolog. (b) Western blot on bacterial lysates to assess the effect of myc-tagged AcrVA2 or control proteins on Cas12a-3xHA expression. 33362, 237, and 58069 refer to the Moraxella bovoculi strains that encode each Cas12a ortholog. RNAP, RNA polymerase (loading control). (c) Codon-modified mbcas12a mRNA levels were quantified by qRT-PCR and normalized to rpoD. Expression is shown relative to empty vector control using the ΔΔCt method. Points represent technical triplicates (n = 3 technical replicates) from one representative experiment; mean ± standard deviation indicated. (d) Western blot on bacterial lysates to assess downregulation of codon-modified HA-tagged MbCas12a by myc-tagged AcrVA2 and AcrVA2.1 relative to controls. RNAP, RNA polymerase (loading control). Experiments in (a–d) were independently repeated at least twice with similar results.
Extended Data Fig. 2 Comparative transcriptomic analysis of bacteria expressing AcrVA2 and controls.
(a–d) Gene expression was quantified as reads per kilobase per million mapped reads (RPKM), normalized to the housekeeping gene rpoD, and log2-transformed. Each point represents one gene. The diagonal line denotes equal expression between samples. (e) Raw read coverage is shown across the full mbcas12a open reading frame, with read depth indicating transcript abundance at each position. The mapping was performed using standard RNA-seq alignment workflows, and raw coverage profiles are presented without normalization to highlight differences in expression levels between samples. Reads Per Kilobase of transcript per Million mapped reads (RPKM) values for mbcas12a are as indicated for each sample: AcrIIA4, 217; AcrVA1, 316; AcrVA2, 38; AcrVA2 H286A, 225; AcrVA2.1, 71. Samples were processed simultaneously, and the experiment was not repeated.
Extended Data Fig. 3 AcrVA2 does not inhibit or downregulate cas12a in vitro.
(a) In vitro cleavage assay with purified anti-CRISPR protein added to MbCas12a(33362) ribonucleoprotein complexes programmed to target a double-stranded DNA amplicon. Cas12a and crRNA were assembled into ribonucleoprotein complexes before addition of anti-CRISPR or control. Anti-CRISPR or control protein was added at a 1:10 or 1:1 ratio relative to Cas12a. (b) In vitro cleavage assay with purified anti-CRISPR protein added to apoMbCas12a(33362) before addition of crRNA and target dsDNA. Anti-CRISPR or control protein was added in a 10:1 or 1:1 ratio relative to apoCas12a. (c) Phage plaque assay using ten-fold serial dilution of phage to assess Cas12a inhibition by 10xHis-MBP-AcrVA2 relative to AcrVA2 and vector control. (d) Phage plaque assay on strains expressing different homologs of mbcas12a from M. bovoculi strains 237 and 33362. Ten-fold serial dilutions of phage were plated on bacterial lawns to assess Cas12a inhibition. (e) Western blot on bacterial lysates to assess downregulation of different mbcas12 orthologs by AcrVA2 E98A/D129A/D195A relative to controls. Experiments in (a–e) were independently repeated at least twice with similar results.
Extended Data Fig. 4 Fusing the N-terminal polypeptide of Cas12a to other proteins enables their downregulation.
(a) mRNA levels were quantified for cas12a-rfp fusions and normalized to rpoD. Numbers refer to the amino acid length of Cas12a fused to RFP-3xHA. Expression is shown relative to samples encoding full-length cas12a with acrIIA4 control using the ΔΔCt method. (b) mRNA levels were quantified for cas12a-cas9 fusions by qRT-PCR and normalized to rpoD. Expression is shown relative to samples with cas9 empty vector control using the ΔΔCt method. For both (a) and (b), points represent technical triplicates (n = 3 technical replicates) from one representative qRT-PCR experiment; mean ± standard deviation indicated. (c) Western blot on bacterial lysates to assess effect of AcrVA2-myc or controls on Cas12a-Cas12f fusions. For (a–c), numbers indicate length of truncated Cas12a polypeptide in amino acids. RpoD, loading control. All experiments were independently repeated at least twice with similar results.
Extended Data Fig. 5 Cas12a N-terminal amino acids are required for mRNA degradation by AcrVA2.
(a) mRNA levels were quantified for wildtype cas12a and mutant cas12a lacking the first 30 amino acids (but provided with a start codon) and normalized to rpoD. Expression is shown relative to acrIIA4 control. (b) RNA levels were quantified for wildtype and mutant cas12a and normalized to rpoD. Expression is shown relative to rfp control. For both (a) and (b), points represent technical triplicates (n = 3 technical replicates) from one representative qRT-PCR experiment calculated using the ΔΔCt method; mean ± standard deviation indicated. Each experiment was independently repeated at least twice with similar results.
Extended Data Fig. 6 Predicted structures of AcrVA2 and Cas12a polypeptide.
(a) Predicted AlphaFold structures of AcrVA2 (teal) with 30 amino acid polypeptides (yellow) from Moraxella bovoculi strains 237 and 33362 and Lachnospiraceae bacterium ND2006. LSKT amino acids (red) in Cas12a and H286, R288, and H291 (purple) in AcrVA2 are indicated. (b) Polypeptide sequences of Cas12a from Moraxella bovoculi strains 237 and 33362 and Lachnospiraceae bacterium ND2006.
Extended Data Fig. 7 AcrVA2 co-fractionates with ribosomes and polysomes independently of Cas12a.
(a) Sedimentation profiles and protein analysis were performed on lysate samples (1–20) collected after separation on a sucrose gradient. Bacteria were engineered and induced to co-express 3xHA-MbCas12a(299aa)-SecM with AcrVA2-myc or AcrVA2H286A-myc. Western blots were performed on samples using anti-Myc and anti-HA antibodies. Coomassie G-250 was used to stain total protein. Images indicate gene expression machinery enriched in different fractions based on A260 traces, total protein stain, and mass spectrometry analysis. (b) Western blot for AcrVA2-myc on fractions (1–20) collected after lysates were separated on a sucrose gradient. Bacteria were engineered and induced to co-express 3xHA-MbCas12a(299aa)LSKT>AAAA-SecM with AcrVA2-myc or AcrVA2H286A-myc. Images indicate gene expression machinery enriched in different fractions. (c) Western blot for AcrVA2-myc on lysate samples (1–20) collected after separation on a sucrose gradient. Bacteria were engineered and induced to co-express 3xHA-SpCas9(299aa)-SecM with AcrVA2-myc or AcrVA2H286A-myc. Images indicate gene expression machinery enriched in different fractions. Experiments were independently repeated at least twice with similar results.
Extended Data Fig. 8 AcrVA2 does not downregulate cas12a directly in vitro.
(a) Purified MBP-AcrVA2 protein or controls were incubated with mbcas12a mRNA in vitro with or without purified MbCas12a(33362) protein added in trans. Samples were resolved on a TBE-urea gel and strained with SYBR gold. AcrIC1 is a type I-C CRISPR-Cas inhibitor that does not inhibit Cas12a. (b) Purified MBP-AcrVA2 or MBP control proteins were added to PURE transcription-translation reactions in which Cas12a(560aa)-RFP-3xHA was expressed from a linear DNA template. Reactions were resolved on SDS-PAGE and probed by Western blot. MBP, maltose binding protein. (c) Western blot on PURE transcription-translation reactions in which Cas12a(560aa)-RFP-3xHA was co-expressed with AcrVA2-myc or GST-myc expressed from linear DNA templates. Glutathione S-transferase (GST) was used as a control. Experiments were independently repeated at least twice with similar results.
Extended Data Fig. 9 RNase E is not required for cas12a mRNA degradation by AcrVA2.
(a) Northern blot on mbcas12a mRNA co-expressed with AcrVA2 and Dip or vector controls in bacteria. Ribosomal RNA (rRNA) included as a loading control. (b) Western blot on bacterial lysates harvested from the same experiment as (a). (c) Growth assay on bacteria expressing MbCas12a and Dip or vector control. Time indicates minutes after subculture and Dip induction. (d) mRNA levels were quantified for pqsB by qRT-PCR and normalized to rpoD. Expression is shown relative to samples with vector control using the ΔΔCt method. Points represent technical triplicates (n = 3 technical replicates) from one representative experiment; mean ± standard deviation indicated. Experiments in (a–d) were independently repeated at least twice with similar results.
Extended Data Fig. 10 AcrVA2 orthologs from other bacterial species do not inhibit Cas12a.
Phage plaque assay on strains expressing different orthologs of Cas12a and AcrVA2. Ten-fold serial dilutions of phage were plated on bacterial lawns to assess Cas12a inhibition. All plaque assays were repeated at least twice with similar results.
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This file contains Supplementary Fig. 1 and Supplementary Tables 1-2. Supplementary Fig. 1: Uncropped images used in main figures and extended data. Black dash boxes indicate areas cropped for the main figure. Supplementary Table 1: Strains used in this study. Supplementary Table 2: Sequences used for protein alignment. Amino acid sequences of AcrVA2 homologs used to generate alignments in Supplementary File 2.
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Marino, N.D., Talaie, A., Gerovac, M. et al. Translation-dependent degradation of cas12 mRNA triggered by an anti-CRISPR. Nature (2026). https://doi.org/10.1038/s41586-026-10440-8
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DOI: https://doi.org/10.1038/s41586-026-10440-8
