Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Abstract

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

Keywords: CRISPR-Cas; Cas9; Cas9 inhibitor; Listeria monocytogenes; anti-CRISPR; bacteriophage; dCas9; gene editing; prophage.

Figure 1. A survey for CRISPR-Cas9 genomic self-targeting (ST) in Listeria monocytogenes

(A) A schematic depicting the principle of genomic self-targeting, where a mobile genetic element (MGE) possesses a target sequence for a spacer in a CRISPR array in the same genome. CRISPR-Cas9 function in this “self-targeting genome” is presumably inactive for continued cell viability. (B) The abundance of genomes with (red) and without (gray) cas9-linked self-targeting (ST), in L. monocytogenes genomes. See Table S1 for a list of self-targeting strains. (C) An example of an ST event, where spacer 16 in the type II-A CRISPR array of strain J0161 has a perfect PAM and protospacer match with a resident prophage (ϕJ0161a). See Figure S1B for the entire CRISPR array.

Figure 2. A prophage from L. monocytogenes J0161 inhibits CRISPR-Cas9 function

(A) The type II-A CRISPR-Cas locus in L. monocytogenes 10403s. Four cas genes and the upstream tracrRNA are indicated, along with a CRISPR array containing 30 spacers. The predicted direction of transcription is indicated with black arrows. Subsequent experiments utilize a non-targeted plasmid (pNT) and a targeted plasmid (pT) that has a protospacer matching spacer 1 in this strain. (B) Representative pictures of colonies of Lmo 10403s wild type (wt), prophage-cured (ϕcure), cas9-deletion strain (Δcas9), and a cas9 overexpression strain (Δcas9 + cas9) after being transformed with pT or pNT plasmids. Bar graphs below the plates show the calculated transformation efficiency (colony forming units per μg of plasmid). Data are represented as the mean of three biological replicates +/− SD. L.D. limit of detection, transformants with small colonies denoted with #. (C) Plasmid-targeting assay with wild type J0161 (contains the ϕJ0161a prophage; experiment conducted as in (B), except with pT_J0161 as the targeted plasmid) is shown in red to denote self-targeting (as in Figure 1). (D) A schematic demonstrating the construction of a 10403s strain containing the prophage ϕJ0161a (10403s::ϕJ0161a). See STAR Methods for details. (E) Plasmid-targeting assay with 10403s lysogenized with the ϕJ0161a prophage (10403s::ϕJ0161a) with endogenous (no mod) or overexpressed cas9 (Δcas9 + cas9; experiment conducted as in (B)).

Figure 3. Identification of four distinct anti-CRISPR proteins

(A) Comparison of the open reading frames from two similar prophages from L. monocytogenes 10403s and J0161. Unique genes (red) comprising ten fragments of ϕJ0161 were tested for CRISPR-Cas9 inhibition in 10403s. n.e., No effect on CRISPR-Cas9 activity, tox., fragment toxic when expressed, t., location of self-targeted protospacer. The encircled fragment exhibited anti-CRISPR activity with two genes (acrAII1, acrAII2) independently capable of inhibiting CRISPR-Cas activity. Conserved (grey) genes were not tested. For reference, phage genes involved in cell lysis, capsid assembly and host integration (int.) are labeled. (B) Representative colony pictures of Lmo 10403s ϕcure strains constitutively expressing “fragment 1” (as shown in (A)) or the indicated individual genes from ϕJ0161a transformed with pNT or pT. The rightmost panels show a 10403s lysogen of ϕJ0161a with CRISPR-Cas9 inhibitor genes deleted (::ϕJ0161aΔacrIIA1-2). See Figure S2 for data from the other ϕJ0161a fragments and Figure S2/S3 for full plates. (C) Representative colony pictures of Lmo 10403s ϕcure strains constitutively expressing acrIIA3, acrIIA4, or orfD transformed with pNT or pT.

Figure 4. Genomic organization and prevalence of acrIIA genes

(A) The genomic context of acrIIA1 (1) and its homolog from L. monocytogenes (orfD) are depicted to scale as cartoons with acrIIA1 homologs in vertical alignment. Typically, acrIIA genes are encoded within prophages adjacent to or near the phage lysin (ply) gene. Genomic neighbors of acrIIA1 and orfD (acrIIA1-4, orfA-E) are shown. Individual genes (***) were assayed for CRISPR-Cas9 inhibition in L. monocytogenes 10403s (see Figure 3 and Figure S3). Helix-turn-helix (HTH) and AP2 DNA binding motifs were detected in some proteins using hidden markov model (HMM) prediction software (Söding et al., 2005). (B) Pie-graph representation of the frequency of each acrIIA gene co-occurrences (C) Pie-graph representation of the prevalence of acrIIA and cas9 genes in the L. monocytogenes pangenome. See Table S1 for relevant accession numbers.

Figure 5. Phylogenetic analysis of AcrIIA1-4 homologs

An unrooted phylogenetic reconstruction of full-length protein sequences identified following an iterative psi-BLASTp search to query all non-redundant protein sequences within GenBank for (A) AcrIIA1. BLASTp was used to identify sequences for similar phylogenetic reconstructions of (B) AcrIIA2, (C) AcrIIA3, (D) AcrIIA4 (see STAR Methods). Selected bootstrapping support values are denoted with filled ovals (≥90%), open rectangles (≥70%) or dashed lines (<70%). The sequence family that is boxed-in represents the family that was tested for anti-CRISPR function. Other homologs reflect distinct sub-families present in the genomes described under the tree.

Figure 6. Inhibition of Streptococcus pyogenes dCas9 and Cas9

(A) A schematic outlining the experimental setup, where single-cell fluorescence of E. coli BW25113 expressing Streptococcus pyogenes (Spy) dCas9 and a sgRNA targeted towards a chromosomal red fluorescent protein (RFP) gene was measured by flow cytometry. (B) Candidate (orf) and validated (acr) acrIIA genes were tested for their ability to inhibit dCas9-based gene repression. Measurements taken reflect the median RFP fluorescence value of a single cell in a unimodal population normalized for each candidate gene to a sgRNA-free control. Error bars represent the mean +/− SD of at least three biological replicates. See Figure 3 and Figure S3 for gene-identification information. See Figure S4 for raw flow cytometry data. (C) A schematic outlining the experimental setup, where HEK293T cells with a chromosomally-integrated, doxycycline-inducible eGFP cassette were transfected with a plasmid encoding a single transcript tracrRNA/eGFP-targeting sgRNA and NLS-SpyCas9 alongside expression constructs encoding one of five codon-optimized phage genes at different ratios. The percent of eGFP positive cells was measured 12 hours after induction by flow cytometry. (D) Candidate (orf) and validated (acr) acrIIA genes were tested for their ability to inhibit dCas9-based gene editing. An increasing amount of inhibitor plasmid (in ng) was added from left to right, at a ratio to the Cas9/sgRNA plasmid of 1:1 and 3:1. Data were normalized to transfection with no phage ORF as the baseline. Average percent of eGFP positive cells is depicted +/− SD across biological triplicates. See Figure S5 for raw flow cytometry data.