RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry

Abstract

To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.

Fig. 1.. A small noncoding RNA (rAcrVIA1) inhibits type VI CRISPR interference.

(A) Schematic of genetic locus encoding AcrVIA2 (orange) and rAcrVIA1 (blue) found in integrated plasmid of L. seeligeri strain LS21. Predicted promoter −10 and −35 elements driving rAcrVIA1 transcription. (B) Plasmid targeting assay demonstrating Cas13 inhibition. Conjugative plasmids expressing a Cas13 target (or not) were introduced into L. seeligeri strains harboring the indicated Acr constructs. Black arrows and lines indicate nonsense mutations. Numbers indicate number of acrVIA2 coding DNA sequence (CDS) nucleotides left after truncation. Quantification of three biological replicates is shown. T/NT, ratio of transconjugants observed with target plasmid to that with nontarget plasmid. (C) Small RNA (sRNA) sequencing reads mapped to the racrVIA1 locus when expressing racrVIA1-acrVIA2 in strain LS1. Combined reads from two biological replicates are shown. (D) Northern blots using probe antisense to racrVIA1 or 5S ribosomal RNA (rRNA).

Fig. 2.. Secondary structure, processing, and Cas13-binding activity of rAcrVIA1.

(A) Predicted secondary structure of rAcrVIA1, with SLs (SL1 and SL2). Red nucleotides indicate positions mutated in (B). (B) Plasmid-targeting assay demonstrating that mutations in each SL abolish Cas13 inhibition, whereas compensatory mutations that restore complementary rescue inhibition. Quantitation of three biological replicates. (C) Denaturing gel analysis of RNA associated with the indicated Cas13 allele in the presence or absence of rAcrVIA1. RNA molecular weight markers in nucleotides. (D) sRNA sequencing reads mapping to racrVIA1 locus from RNA extracted from purified apod-Cas13 in the presence of rAcrVIA1. IP, immunoprecipitation.

Fig. 3.. Overall architecture of the Cas13-rAcrVIA1 complex.

(A) Domain organization of Cas13. (B) Surface (left) and ribbon (right) representation of the cryo-EM structure of Cas13-rAcrVIA1 complex in different orientations. Domains are colored as in (A). (C) Ribbon representation (left) and schematic drawing (right) of the sequences and secondary structure of rAcrVIA1. Segments that could be traced are in color, and disordered segments are in gray. (D) Structural comparison of Cas13-bound rAcrVIA1 (red) and crRNA (gray). The repeat and spacer regions of the crRNA are colored in black and gray, respectively. (E) Structural comparison of Cas13-rAcrVIA1 complex and the Cas13-crRNA complex. The vector length correlates with the scale of domain movement. Domains are colored as in (A). (F) Comparison of the positions of the four catalytic residues from HEPN-I and HEPN-II domains between rAcrVIA1-bound and crRNA-bound Cas13. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 4.. rAcrVIA1 acts independently of AcrVIA2 and is regulated by ORF1.

(A) Schematic of genetic locus encoding HTH-containing ORF1, rAcrVIA1, and AcrVIA2. (B) (Top) Analysis of dCas13-associated RNA in presence of the indicated Acrs. (Bottom) dCas13-his6 Western blot. (C) Plasmid-targeting assay in which plasmids encoding Acrs were directly targeted by preexisting Cas13 RNP complexes. Acr plasmids were conjugated into strain LS1 with or without a targeting spacer, and transconjugants were enumerated. T/NT, targeted to nontargeted transconjugants ratio. Quantification of three biological replicates. Asterisks indicate statistical significance (P < 0.05, Student’s t test). ns, not significant. (D) RNA cleavage assay with purified 3xFlag-tagged Cas13-crRNA and Cy3-labeled target RNA substrate, with rAcrVIA1 added in 1:8, 1:2, and 2:1 molar ratios. Representative of three biological replicates. (E) Plasmid-targeting assay. Conjugative plasmids expressing a Cas13 target (or not) were introduced into L. seeligeri strains harboring the indicated Acr constructs. Quantification of three biological replicates. (F) Transcriptional activity of the indicated promoters in the presence and absence of ORF1. RFU, relative fluorescence units. (G) Northern blots using probes antisense to rAcrVIA1 or 5S rRNA sequence.