Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

Abstract

CRISPR–Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR–Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16–19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9–Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR–Cas9 inhibition.

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Fig. 1: Identification of four type II-A Cas9 inhibitors, AcrIIA16–19.
Fig. 2: Prevalence of acrIIA genes in integrative MGEs and their effect on CRISPR targeting during conjugation.
Fig. 3: In vitro binding and inhibition activities of AcrIIA16–19 against SpyCas9.
Fig. 4: In vivo co-immunoprecipitation of AcrIIA16–19 and SpyCas9.
Fig. 5: Acr-mediated inhibition of Cas9 orthologues during gene editing in human cells.

Data availability

All data generated or analysed during this study are included in this published article and its Extended Data, Source Data and Supplementary Information files. All relevant accession codes are available in Table 1.

Change history

  • 23 April 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  • 30 April 2020

    In the version of this Article originally published, Extended Data Fig. 4 was missing a label for panel c; this has now been corrected.

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Acknowledgements

We thank K. L. Palmer (UT Dallas) and G. M. Dunny (University of Minnesota) for providing all E. faecalis strains, vector and protocols. We thank M. Johnson for his assistance in executing PlasFlow, N. Marino for her profound wisdom and comedic wit, and all members of the Bondy-Denomy Lab for their continuous support and thoughtful discussions contributing to this study. Bondy-Denomy Lab was supported by the UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation, the Searle Fellowship, the Vallee Foundation, an NIH Director’s Early Independence Award No. DP5-OD021344, and NIH grant no. R01GM127489. Research in the Kleinstiver Lab is supported by NIH grant no. R00-CA218870, an A.S.G.C.T. Career Development Award and the Margaret Q. Landenberger Research Foundation. This research was carried out using Defense Advanced Research Projects Agency (DARPA) award HR0011-17-2-0043. The views, opinions and/or findings should be attributed to the author and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government.

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Authors

Contributions

C.M. and J.B.-D. conceived and designed the study. C.M. conducted bioinformatic, bacterial and biochemical experiments. K.A.C. performed human cell experiments. B.A.O. and R.P.-R. assisted with experimental design. B.P.K and J.B.-D. supervised experiments. C.M. and J.B.-D. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Joseph Bondy-Denomy.

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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. B.P.K. is an inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies, is a consultant for Avectas Inc. and an advisor for Acrigen Biosciences.

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Extended data

Extended Data Fig. 1 Schematic of acr loci and lethal self-genome cleavage assay.

a, Full schematic of acr loci with relevant neighboring genes displayed. b, Schematic of SpyCas9 in P. aeruginosa programmed to cause lethal self-genome cleavage to assess bacterial survival in the presence of AcrIIA proteins. CRISPR strength is determined by titrating levels of IPTG, which induces expression of sgRNA targeting the chromosomal phzM gene from a multicopy plasmid. OD600 measurements are represented as the mean of three biological replicates ± SD.

Extended Data Fig. 2 Anti-CRISPR distribution in integrative mobile genetic elements across bacterial taxa.

Phylogenetic analysis based on acrIIA16–19 homologs (panels a to d, respectively) reconstructed from a midpoint rooted minimum-evolution of full length protein sequences identified following an iterative PSI-BLASTp search, see methods for details. Number of genomes included to construct each tree for acrIIA16–19 are seventy, twenty-six, eighty-four and seventeen respectively. Branches are labeled with species name and colored according to species class (see legend). Species for which AcrIIA homologs have been tested in this study are shown in bold.

Extended Data Fig. 3 AcrIIA enhance conjugation-mediated horizontal gene transfer in E. faecalis, related to Fig. 2.

a, b. Mating outcomes during plasmid conjugation of a targeted plasmid from donor to recipient cells where indicated acrIIA genes are (a) pre-expressed in recipient cells, or (b) encoded on conjugating plasmid. Data displayed as 10-fold colony serial dilution spots of donor, recipient or transconjugant cells on selective antibiotic plates. Mating assays were performed in biological triplicate and produced similar outcomes. c, Schematic of E. faecalis conjugation of protospacer and acrIIA-bearing plasmid transferring into CRISPR-defective recipients. For CRISPR1, the bona fide AcrIIA4 is utilized to suppress CRISPR targeting, and a ΔCas9 strain from previously reported work is used for CRISPR3 (Price et al.,5). Red * denotes plasmids that have lost conjugation ability. Mating assays were performed in biological duplicate or triplicate and produced similar outcomes.

Extended Data Fig. 4 AcrIIA16–19 biochemical analysis, related to Figs. 3 and 4.

a, Coomassie-stained polyacrylamide gel showing GST-tagged AcrIIA proteins (IIA4 37kD, IIA16Lmo 50kD, IIA17Sga 39kD, IIA18Sma 48kD and IIA19Ssim 42kD) purified from E. coli by elution from Glutathione Sepharose columns. Visible bands at different sizes are co-purifying proteins from E. coli. Data shown are representative of two independent experiments. b, Coomassie-stained polyacrylamide gel showing co-immunoprecipitation of Acr proteins with Myc-tagged sgRNA-bound SpyCas9 pulled down from P. aeruginosa. Data shown are representative of two independent experiments. c, d, Uncropped versions of both Myc and GST pulldowns from Fig. 4a and c, displaying all fragments of (c) sgRNA-bound SpyCas9, or (d) Apo- SpyCas9 without sgRNA present. Data shown are representative of two independent experiments. Source data

Extended Data Fig. 5 Acr inhibition activity in human cells tested against different Cas9 orthologs, related to Fig. 5.

Reported Acr proteins in this study and from previous works tested for inhibition of genome editing activities of Sth1Cas9, Sth3Cas9, Nme2Cas9 and CjeCas9 (a-d, respectively). Editing efficiencies against endogenous genes in HEK 293 T cells were assessed by targeted sequencing and quantified as the percentage of reads containing a nuclease-induced alteration; the no-Acr condition contains an EGFP expression plasmid; the NT control includes an empty U6 expression plasmid. Percent reads modified are represented as the mean of three biological replicates ± SD. Source data

Supplementary information

Supplementary Information

Supplementary Information for Extended Data 2 phylogenetic tree analyses. Raw versions of the trees in NEXUS format.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–4.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed gels and northern blots.

Source Data Fig. 4

Unprocessed gels and western blots.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed gels and western blots.

Source Data Extended Data Fig. 5

Statistical source data.

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Mahendra, C., Christie, K.A., Osuna, B.A. et al. Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nat Microbiol 5, 620–629 (2020). https://doi.org/10.1038/s41564-020-0692-2

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