Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Abstract

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.

Keywords: CRISPR; Cas9; anti-CRISPR; genome editing; self-targeting.

Fig. 1.

Identification of self-targeting Staphylococcus strains that contain active type II CRISPR-Cas systems. (A) Acquisition of protospacers from MGEs can result in self-targeting if the MGE is capable of stably integrating (e.g., prophages) or associating (e.g., plasmid) with the genome. (B) Overview of TXTL assay to test for Acr activity. Cas9 template DNA is combined with an sfGFP reporter plasmid and a plasmid expressing a sgRNA targeting the reporter. In the absence of Acr activity, expression of the Cas9 RNP suppresses sfGFP expression. In the presence of Acrs, the Cas9 RNP is inhibited, resulting in increased fluorescence. (C) Genomic amplicons from S. schleiferi and S. haemolyticus containing Cas9 lower sfGFP expression with an sfGFP-targeting sgRNA, demonstrating that the natural CRISPR loci are active.

Fig. 2.

Identification of three Acrs using amplicon screening and guilt by association. (A) The relative level of S. schleiferi (Left) or S. haemolyticus (Right) Cas9 DNA cleavage inhibition for each fragment was measured as percentage of the GFP expression for the nontargeting (NT) control after subtracting the fluorescence level observed in the targeting (T) control with no Acr present. Thus, 0% inhibition is equivalent to the GFP expression level measured with the targeting sgRNA, while 100% inhibition represents no reduction in the maximum GFP expression. GF5 is the only amplicon that exhibits Acr activity. (B) Genes found in GF5. (C) Each gene in GF5 was individually cloned and tested for Acr activity with TXTL and SauCas9. The second gene of GF5 (AcrIIA13) inhibits SauCas9. (D) AcrIIA13a, a homolog of AcrIIA13, is found in GF8 and is also able to inhibit SauCas9 in a TXTL assay. GF8, which contains AcrIIA13a, did not exhibit Acr activity, however. (E) Of 10 candidates chosen from a guilt-by-association search seeded by GF5 gene 1, candidates 1 (AcrIIA14) and 10 (AcrIIA15) were found to inhibit SauCas9 using the TXTL assay. All data in this figure are from single TXTL runs.

Fig. 3.

Three SauCas9 inhibitors with distinct features and activities. (A) Plot showing inhibition of SauCas9 cleavage activity by AcrIIA13, AcrIIA14, and AcrIIA15 with %cleavage plotted on y axis and Acr concentration (nanomolar) on the x axis. (Left) Plot corresponding to the cleavage assays performed by first complexing Cas9 and sgRNA followed by addition of Acr and DNA. (Right) Plot corresponding to the cleavage assays performed by first complexing Cas9 with Acrs followed by the addition of sgRNA and target dsDNA. The plotted data are the average %cleavage activity from three independent replicates. Error bars represent average ± SD. (B) Six percent polyacrylamide gel showing formation of Cas9-sgRNA RNP in the presence and absence of different Acrs. Lanes boxed in red represent reactions where Acrs were added before the addition of sgRNA. Lanes boxed in blue represent reactions where Acrs were added after the addition of sgRNA. Order of the addition of different reaction components are shown above the boxes. (C) Six percent polyacrylamide gel showing binding of Cas9-sgRNA RNP to either target DNA (T) or nontarget DNA (NT) in the presence and absence of different Acrs. Lanes boxed in red represent reactions where Acrs were added to Cas9 before the addition of sgRNA and dsDNA. Lanes boxed in blue represent reactions where Acrs were added after the addition of dsDNA to Cas9-sgRNA RNP. Order of the addition of different reaction components are shown above the boxes. (D) Agarose gels showing cleavage of dsDNA target by SauCas9 in the presence of increasing amounts of each Acr with the N terminus removed. Reactions were performed by adding Acrs after complexing Cas9 and sgRNA (Top) or prior to the addition of sgRNA (Bottom), as indicated to the Left of each panel. Each reaction contains 5 nM dsDNA, 100 nM SauCas9, 100 nM sgRNA, and the following Acr concentrations: 50 nM, 100 nM, 200 nM, 500 nM, 1,000 nM, and 2,000 nM. Uncleaved and cleaved dsDNA products are indicated by green and blue arrows, respectively.

Fig. 4.

AcrIIA13, AcrIIA14, and AcrIIA15 share an N-terminal domain that binds a set of inverted repeats proximal to their promoters. (A) Schematic showing the gene structure of AcrIIA13, AcrIIA14, and AcrIIA15. The gene section highlighted in blue is highly conserved across the different Acrs and contains the conserved HTH motif. The C-terminal region shows high variability across the three Acrs. (B) Promoter-proximal sequences for AcrIIA13–AcrIIA15 showing the presence of two sets of IRs. IR segments are highlighted in yellow with the inverted repeat sequence underlined. Schematic outlines hypothesis that the HTH motif within the Acrs binds to the promoter-proximal sequence spanning inverted repeats IR1 and IR2 to self-regulate Acr transcription. (C) Sequence of the dsDNA substrates used for EMSAs in D and E. These sequences are encoded within the promoter-proximal region of AcrIIA15 that were either synthesized in its wild-type form or mutated to serve as negative control. (D) Individually purified Acr proteins bind to the AcrIIA15 promoter-proximal sequence containing inverted repeats, causing an upshift. (E) AcrIIA15 does not bind to a mutated form of the AcrIIA15 IR dsDNA substrate (Left). Removing the N-terminal domain from AcrIIA15 prevents binding to the wild-type (WT) IR DNA (Right).

Fig. 5.

AcrIIA13 is a potent and selective inhibitor of SauCas9 in mammalian cells. (A) Schematic of a mammalian validation platform to express Cas9, sgRNAs, and an Acr protein from different stably integrated lentiviral vectors in human cells, to quantify the CRISPR-Cas genome editing inhibitory potential of the Acr candidates by flow cytometry. HEK-RT1 cells, a human HEK293T-based genome editing reporter cell line with a doxycycline-inducible GFP, were sequentially transduced with lentiviral vectors expressing Acr candidates, SauCas9 or SpyCas9, and guide RNAs targeting GFP or a negative control. Three days posttransduction of the guide RNAs, GFP expression was induced by treatment with doxycycline for 24 h. Genome editing inhibition efficiency for each Acr candidate was measured as the increase in percentage GFP-positive cells expressing on-target guide RNAs (sgGFP, mCherry-positive), compared to controls. (B) Quantification of SauCas9 and SpyCas9 inhibition efficiency of AcrIIA13, an N-terminal truncation of AcrIIA13, and an AcrIIA13 homolog (AcrIIA13b) in human cells. Error bars represent the SD of triplicates. sgC, sgGFP1/sgGFP8: negative control and GFP-targeting guide RNAs for the respective Cas9s. The data shown for AcrIIA13 are partial quantification of raw flow cytometry data shown in SI Appendix, Fig. S8. (C) Assessment of SauCas9 and SpyCas9 inhibition by AcrIIA14, an N-terminal truncation of AcrIIA14, AcrIIA15, and an N-terminal truncation of AcrIIA15, in human cells. The assay was run as described above.