A scoutRNA Is Required for Some Type V CRISPR-Cas Systems

Abstract

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Keywords: CRISPR-cas; Candidate Phyla Radiation (CPR) bacteria; Cas12c (C2c3); Cas12d (CasY); RuvC nuclease domain; crRNA; scoutRNA; tracrRNA.

Figure 1 |. Cas12c/d are part of compact CRISPR systems found in tiny genomes.

A) Diagram of Type V-C and Type V-D CRISPR-Cas loci. Cas12c (C2c3) and Cas12d (CasY) that share minimal sequence similarity with Cas12a (Cpf1) except for the RuvC catalytic domain. B) Unrooted phylogentic tree showing Cas12c and Cas12d representatives. Newly identified orthologs are highlighted with colored circles (orange, Cas12c; blue, Cas12d) and greyed out circles mark previously described orthologs. Orthlogs used for experiment’s in this study are identified by name. C) Host assignment for all CRISPR systems, Cas12c and Cas12d illustrating that Cas12d is highly enriched in Candidate Phyla Radiation (CPR) bacteria. D) A plasmid depletion screen for PAM-dependent inhibition of plasmid transformation showing that only target sequences adjacent to a TR sequence were efficiently depleted. E) Plasmid interference against individual PAM targets showing clearance of plasmids containing a TA or TG adjacent to the targeted sequence. F) Predicted number of sites in a CPR-associated bacteriophage genome that are targetable by Cas12a, Cas12e and Cas12d.

Figure 2 |. Cas 12c/d requires a new kind of tracrRNA for DNA interference.

A) Plasmid transformation assay testing RNA-guided DNA targeting by CRISPR-Cas systems expressed in E. coli. Deletions were made of non-coding regions of the CRISPR locus and resulting plasmid transformation efficiencies are shown. B) Diagram of CRISPR-Cas12c genomic loci indicating a noncoding sequence between the cas1 and cas12c genes; Northern blot using a radiolabeled DNA oligonucleotide probe (represented by red arrow) and affinity-purified samples of Cas12c when co-expressed with noncoding regions of the CRISPR locus, (IVT, in-vitro transcribed; KO, knockout). C, D) RNA-sequencing data corresponding to the CRISPR-Cas non-coding locus, from samples that were affinity purified from E. coli expression (C) or obtained from metatranscriptomic analysis (D). Black diamonds in CRISPR loci cartoons represent repeats and white rectangles represent spacers. Purple rectangles correspond to the non-coding region and the predicted secondary structure of this region is shown to the right. Color scale represents base-pair probabilities.

Figure 3 |. cis- and trans-cleavage activities of Cas12d Cas12d-catalyzed and crRNA-targeted DNA cleavage.

A) ScoutRNA is essential for Cas12d-mediated dsDNA cleavage. In this assay, nontarget strand is 5’-end labeled, and the reactions were conducted in the absence (−) or presence (+) of scoutRNA. B) Time course plots of cis-cleavage activity of Cas12d. C) Time course plots of trans-cleavage activity of Cas12d. The substrates of dsDNA, ssDNA and ssRNA used in this assay are non-specific to Cas12d crRNA. D) Cas12d cleavage activities on mutated dsDNA targets. In this assay, pairs of mismatched base pairs were tiled across the crRNA-target DNA strand duplex, and the resulting extent of crRNA-guided Cas12d-catalyzed dsDNA cleavage is shown.

Figure 4 |. A short conserved sequence in scoutRNA is required for dsDNA targeting.

A) Cas12d-associated crRNA repeat sequence alignment. Conserved sequences are shown in black; predicted scoutRNA secondary structure and possible short base paired interaction between scoutRNA and crRNA repeat are also shown. B) Cas12d strongly binds to the complex from scoutRNA and crRNA. Data are from nitrocellulose filter binding assays with radiolabeled crRNA and/or scoutRNA as a function of Cas12d protein concentration; (*) indicates radiolabeled species when two RNAs were present in the binding reaction. C) The effect of reciprocal changes in guide RNA stem on Cas12d-mediated dsDNA cleavage. wt= wild-type and mut=mutation. D) Importance of 5 conserved nucleotides in Cas12d scoutRNA. Mutants #4 and #5 contained sequence changes that maintained base pairing complementarity in the regions shown; mutant #2 contained nucleotide changes to create a complementary sequence on the strand opposite the conserved 5 nt. sequence.

Figure 5 |. An RNase III-independent dual RNA-guided pre-crRNA processing mechanism.

A) Timecourses of pre-crRNA cleavage in the presence or absence of purified Cas12c and scoutRNA, using a 5’-end radiolabeled 58-nt. pre-crRNA. B) Kinetics of scoutRNA-dependent Cas12c-catalyzed pre-crRNA cleavage using the pre-crRNA substrates shown.

Figure 6 |. Three different types of RNA-guided CRISPR-Cas families defined by RNA components.

Non-coding RNAs enable functional classification of CRISPR-Cas enzymes into three distinct categories. All use crRNA, whereas a subset use either a canonical trans-activating CRISPR RNA (tracrRNA) and another subset use a short-complementarity untranslated RNA (scoutRNA).