A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity

Abstract

The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.

Figure 1.. AcrVIA1 inhibits type VI-A CRISPR-Cas immunity against plasmids and phages.

(A) Transfer of a conjugative plasmid with or without the spc4 target of the L. seeligeri type VI-A CRISPR-Cas system into different strains: wild-type (WT), Δspc Δcas13a or WT harboring the ϕLS46 or ϕLS46 ΔacrVIA1 prophages. (B) Schematic of the ϕLS46 genome showing the four main transcription units (acr, lysogeny, early- and late-lytic genes). gp2 was renamed acrVIA1. The locations of the targets of spacers used in this study are shown in grey. (C) Same as (A) but using strains carrying plasmids to express different acr genes from ϕLS46. (D) Detection of phage propagation after spotting ten-fold dilutions of WT, Δgp1-4 or ΔacrVIA1 phage ϕLS46, on lawns of L. seeligeri ΔRM Δspc or ΔRM ΩspcE2. (E) Same as (D) but spotting ϕLS59 into lawns of L. seeligeri ΔRM Δspc, ΔRM Ωspc59 or ΔRM Ωspc59/pgp2. (F) Growth of WT, Δspc and WT/pgp2 L. seeligeri strains expressing a spc4 target RNA under the control of an anhydrotetracycline-inducible promoter, after addition of the inducer.

Figure 2.. AcrVIA1 interacts with Cas13a^cRNA to prevent binding of the target RNA and RNase activation.

(A) cis-RNA cleavage time course with purified L. seeligeri Cas13a-His6, AcrVIA1 and/or AcrVIA1-3xFLAG using radiolabeled non-target or spc2-target RNA substrates. Reactions were analyzed after 5, 10, or 20 minutes of incubation. (B) trans-RNA cleavage time course as in (A) but using a radiolabeled non-target RNA substrate in the presence of unlabeled non-target or spc2-target RNA. (C) Dose response of Cas13a cis-RNase inhibition by AcrVIA1-3xFLAG (D) Anti-FLAG immunoprecipitation using protein extracts from L. seeligeri cells expressing either Cas13a-His6 alone or co-expressing AcrVIA1-3xFLAG. The His6 and FLAG epitopes, as well as the σ^A protein were detected via immunoblot. (E) EMSA of radiolabeled non-target or spc2-target RNAs in the presence of dCas13a-His6, with 2:1, 1:1, or 1:2 equivalents of AcrVIA1-3xFLAG.

Figure 3.. Cryo-EM structures of Cas13a^crRNA and AcrVIA1-Cas13a^crRNA complexes.

(A) Domain organization of L. seeligeri Cas13a. (B) Schematic representation of the crRNA sequence. The repeat and spacer regions within crRNA are shown in black and red, respectively. The disordered region is shown in gray. The black arrow shows the cleavage site of the pre-crRNA. Inset: crRNA maturation pathway; repeats are represented as “R”, spacers as numbers. (C) Ribbon representation of the structure of Cas13a^crRNA. (D) Ribbon and surface (AcrVIA1) representations of AcrVIA1-Cas13a^crRNA complex. (E-H) Detailed interactions between AcrVIA1 and Cas13a^crRNA in the complex. (I) Transfer of conjugative plasmid with or without spc4 target of the L. seeligeri type VI CRISPR-Cas system into WT L. seeligeri harboring plasmid-borne wild-type or mutant alleles of acrVIA1-3xflag. (J) Anti-Flag immunoblot of AcrVIA1 mutants tested in (I), and anti-σ^A loading control, *cross-reacting protein.

Figure 4.. AcrVIA1 enables full phage escape from type VIA CRISPR-Cas immunity.

(A) Efficiency of plaquing (relative to the number of plaques formed in lawns of L. seeligeri ΔRM Δspc) of phages ϕLS46 or ϕLS46 ΔacrVIA1 in lawns of bacteria expressing spcA1, spcE1, spcE2 or all three (3 spc). Error bars represent SEM from 3 biological replicates. (B-F) Growth of L. seeligeri ΔRM Δspc (B), ΔRM ΩspcE1 (C), ΔRM ΩspcE2 (D), ΔRM ΩspcA1 (E) and ΔRM Ω3spc (F), measured as OD₆₀₀ over time, infected with ϕLS46 or ϕLS46 ΔacrVIA1 phages, or uninfected. The average curves of three different replicates are reported, with +/− SEM values shown.