The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages1,2,3. We identified the first examples of proteins produced by phages that inhibit a CRISPR–Cas system4. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR–Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR–Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase–nuclease and preventing its recruitment to the DNA-bound CRISPR–Cas complex. In vivo, this anti-CRISPR can convert the CRISPR–Cas system into a transcriptional repressor, providing the first example—to our knowledge—of modulation of CRISPR–Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR–Cas function.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007)
Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011)
Jore, M. M., Brouns, S. J. J. & van der Oost, J. RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements. Cold Spring Harb. Perspect. Biol. 4, a003657 (2012)
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)
van der Oost, J., Westra, E. R., Jackson, R. N. & Wiedenheft, B. Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nature Rev. Microbiol. 12, 479–492 (2014)
Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010)
Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011)
van Duijn, E. et al. Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (CRISPR)-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa. Mol. Cell. Proteomics 11, 1430–1441 (2012)
Westra, E. R. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46, 595–605 (2012)
Huo, Y. et al. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nature Struct. Mol. Biol. 21, 771–777 (2014)
Rollins, M. F., Schuman, J. T., Paulus, K., Bukhari, H. S. T. & Wiedenheft, B. Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa. Nucleic Acids Res. 43, 2216–2222 (2015)
Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011)
Wurtzel, O. et al. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog. 8, e1002945 (2012)
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)
Rath, D., Amlinger, L., Hoekzema, M., Devulapally, P. R. & Lundgren, M. Efficient programmable gene silencing by Cascade. Nucleic Acids Res. 43, 237–246 (2015)
Cady, K. C. & O’Toole, G. A. Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J. Bacteriol. 193, 3433–3445 (2011)
Jackson, R. N. et al. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345, 1473–1479 (2014)
Mulepati, S., Héroux, A. & Bailey, S. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science 345, 1479–1484 (2014)
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)
Westra, E. R., Buckling, A. & Fineran, P. C. CRISPR–Cas systems: beyond adaptive immunity. Nature Rev. Microbiol. 12, 317–326 (2014)
Cady, K. C., Bondy-Denomy, J., Heussler, G. E., Davidson, A. R. & O’Toole, G. A. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194, 5728–5738 (2012)
We thank W. Navarre and E. Westra for reading the manuscript. This work was supported by an Operating Grant to A.R.D. (MOP-130482) and to K.L.M. (MOP-136845), both of which were from the Canadian Institutes of Health Research (CIHR). J.B.-D. was supported by a CIHR Canada Graduate Scholarship Doctoral Award and an Ontario Graduate Scholarship award. Research in the Wiedenheft laboratory is supported by the National Institutes of Health (P20GM103500 and R01GM108888), the National Science Foundation EPSCoR (EPS-110134), the M.J. Murdock Charitable Trust, and the Montana State University Agricultural Experimental Station.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Purified Csy complex was fractionated by SEC alone (a) or in the presence of AcrF2 (b). Fractions were analysed on a silver nitrate stained SDS–PAGE gel. The input (IN) and fractions are shown.
a, Cas3 was fractionated by SEC alone or in the presence of AcrF3 or AcrF1. Overlays of plots of elution volume versus optical density at 280 nm of the column eluates are shown. The numbers represent the fractions that were selected for analysis. b–e, Silver nitrate stained SDS–PAGE gels are shown from SEC experiments with Cas3 (b), AcrF3 (c), Cas3 with AcrF3 (d) or Cas3 with AcrF1 (e). The sample that was loaded onto the SEC column is shown as input (In) and fractions from the same elution positions are indicated numerically. AcrF3 is seen eluting in fractions 4–8 only in the presence of Cas3. There is also a visible shift in the Cas3 elution profile in the presence of AcrF3 but not AcrF1 (fractions 3–5).
Isothermal titration calorimetry (ITC) assays showing the Csy complex binding to an 8-nucleotide ssDNA target that comprises the seed region. No binding is observed in the presence of AcrF1, AcrF2 or with a non-target (the reverse complement sequence of the target) ssDNA substrate. A representative run is shown for each condition with the dissociation constant (Kd) value and error of fit from that particular run. Over multiple runs (n = 6) with the Csy complex binding to the ssDNA ligand, the average Kd value was 90 nM ± 37.
The Csy complex was targeted to the promoter of the gene phzM, and repression efficiency was assayed by RT–qPCR (see Methods). The per cent repression of phzM in the indicated strains expressing a phzM-targeting crRNA relative to wild-type (WT) PA14 with an empty plasmid is shown. All values were normalized to rpsL, a gene encoding a ribosomal protein. Means ± s.d. are shown.
Untagged AcrF4 was expressed in E. coli BL21 cells and a crude lysate of these cells was mixed with the Csy complex bound to Ni-NTA beads via a 6×His tag on Csy3. a, The flow through (FT), wash 1 (W1), and two elution fractions (E1, E2) from the Ni-NTA column are shown, as well as a comparison to pure Csy complex. b, The Ni-NTA elution fractions were fractionated by SEC, demonstrating a stable interaction between the Csy complex and AcrF4. The input (In) lane shows the sample that was loaded on the SEC column and numbered fractions are analysed on SDS–PAGE gels.
a, Purified Csy1–Csy2 heterodimer with an MBP and 6×His tag fused to Csy1 was fractionated by SEC in the presence or absence of AcrF1 (boxes indicate the Csy1–Csy2 peak). b, Purified MBP/6×His-tagged Csy3 was fractionated in the presence or absence of AcrF2. These are complementary experiments to those seen in Fig. 3b and c, respectively. Input (In) and selected fractions are shown on SDS–PAGE gels. c, AcrF1 and AcrF2 were incubated with the Csy complex singly or in combination. Asterisks designate which anti-CRISPR was added first to the reactions containing both anti-CRISPR proteins. The addition order did not affect the result since there is no competition for binding sites between these two anti-CRISPR proteins. After incubation, each mixture was fractionated by SEC and the peak Csy complex fraction is shown on an SDS–PAGE gel. In each experiment the anti-CRISPR proteins are in excess relative to the Csy complex.
a, The Csy complex was treated with a low concentration (600 nM, +) of RNase A or a high concentration of RNase A (70 µM, ++). This mixture was fractionated by SEC, revealing Csy4 dissociation at the higher RNase A concentration. Pre-treatment of the Csy complex with RNase A, with the subsequent addition of AcrF1 or AcrF2 followed by SEC fractionation was then conducted. Peak Csy complex fractions are shown on an SDS–PAGE gel. b, A TBE-urea denaturing gel is shown, stained with SYBR gold, showing the native crRNA in the Csy complex and the protected fragments remaining after 70 µM RNase A treatment. c, Quantification of Coomassie blue stained gels from three independent preparations of the respective proteins is shown. Anti-CRISPR proteins bound with unaltered stoichiometry to RNase-A-pre-treated Csy complexes. Error bars represent s.d.
Csy complexes with crRNA molecules possessing spacers of differing lengths (16, 32, or 48 nucleotides) were purified and fractionated by SEC in the presence of AcrF1. A representative Coomassie blue stained SDS–PAGE gel is shown, with twofold dilutions of the peak fraction containing the Csy complex and co-eluting AcrF1. Arrows on the bottom of the gel indicate comparable dilutions based on the levels of Csy1. Note the increasing abundance of Csy3 and AcrF1. b, Lanes with arrows from the gel in a are shown next to each other for comparison.
a, The same samples from Fig. 4a were run on a denaturing TBE-urea gel, stained with SYBR gold, to reveal the crRNA (two species are apparent), and the Csy-complex-bound 50 bp dsDNA. In these experiments, DNA was prebound to the Csy complex, and AcrF1 or AcrF2 were subsequently added to the DNA-saturated Csy complex. This mixture was then fractionated by SEC and the Csy-complex-containing peak fractions were analysed. b, A schematic showing the crRNA sequence with repeat-derived regions shown in black and the variable 32-nucleotide spacer region in red. The seed-interacting region that is critical for target recognition (nucleotides 1–5, 7, 8) is in bold. DNA oligonucleotides used in this study are shown, with labels ‘A’, ‘B’ and ‘C’ corresponding to the targets shown in Fig. 4c. The 8-nucleotide ssDNA substrate was used in ITC experiments (Extended Data Fig. 3), and the 50 bp dsDNA in EMSAs (Figs 1d and 4b).
About this article
Cite this article
Bondy-Denomy, J., Garcia, B., Strum, S. et al. Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins. Nature 526, 136–139 (2015). https://doi.org/10.1038/nature15254
Nature Microbiology (2020)
Biochemical Society Transactions (2020)
Crucial Role of the Accessory Genome in the Evolutionary Trajectory of Acinetobacter baumannii Global Clone 1
Frontiers in Microbiology (2020)
Genome Biology (2020)
Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism
Molecular Cell (2020)