Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs

Abstract

Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8 nucleotide 5' sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicate that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

Figure 1. Cmr2 antibodies immunopurify active Cmr-crRNA complexes

A) Cmr2 antibodies recognize and specifically immunoprecipitate ~100 kD protein. P. furiosus S100 extract (T) and PfCmr2 antibody immune (I) and pre-immune (PI) immunoprecipitation (IP) samples were analyzed by Western blotting with Cmr2 antibodies. Positions of size markers are indicated. ~100 kD protein specifically recognized by immunoblotting and immunoprecipitated by Cmr2 antibodies is indicated by arrow. B) 45- and 39-nt crRNAs are immunopurified with Cmr2 antibodies. RNAs extracted from P. furiosus S100 extract (T) and immune (I) and pre-immune (PI) immunoprecipitation (IP) samples were analyzed by Northern blotting with a probe against P. furiosus crRNA 7.01. The 45- and 39-nt species of 7.01 that are co-IPed with the Cmr2 antibodies are indicated by arrows. Sizes of RNA markers (M) are shown. C) Immunoprecipitated Cmr complexes cleave a target RNA. 5’ end-labeled 7.01 target RNA (complementary to crRNA 7.01, indicated with arrow) was incubated in the absence (−) of proteins or in the presence of immune (I) and pre-immune (PI) immunoprecipitation samples. Cleavage products are marked with asterisks. D) Co-immunopurified crRNAs possess 5’ OH and 3’ phosphate ends. Immunoprecipitated crRNAs were isolated and 5’ or 3’ labeled with (+) and without (−) prior phosphatase treatment. The 45- and 39-nt crRNA species are indicated by arrows and RNA size markers (M) are shown.

Figure 2. Deep sequencing profiles of crRNAs found in total P. furiosus RNA and immunopurified Cmr complexes

A) Total and Cmr-associated small RNA sequencing reads that map to the seven P. furiosus CRISPR loci. Y-axis scale bars indicate the number of reads (thousands) that contain nucleotides that map to the indicated position within the CRISPR locus. Red indicates sequence reads that map to the sense strand relative to the CRISPR leader region, and blue corresponds to reads that map to the antisense strand. The positions of CRISPR repeats are shown as black bars below the graph. 1 kb x-axis scale is indicated. Images were generated using the UCSC archaeal genome browser (Karolchik et al., 2003). B) Putative promoter sequences found in the P. furiosus CRISPR leader regions. Alignment of the regions upstream of the first repeat of each P. furiosus CRISPR locus are shown. The repeats are shown in blue, the initial transcribed regions (detected by sequencing) are shown in red, the potential TATA elements are shown in pink, the potential BRE elements are underlined, and other leader sequences are shown in black. Consensus BRE/TATA elements are shown above the alignment.

Figure 3. Cmr complex crRNAs contain a conserved 8-nucleotide 5’ sequence tag and are of defined 39- and 45-nucleotide lengths

A) The 5’ and 3’ ends of Cmr-associated crRNAs are defined relative to the upstream repeat element. Graphs show the % of crRNAs sequenced in total P. furiosus RNA (upper panel) and immunopurified Cmr complexes (lower) with ends that map at indicated positions relative to the 5’ repeat-guide junction (arrowhead). The Cmr-associated crRNA species are illustrated below with 5’ repeat tag (black) and guide sequence (green) indicated. RNAs from CRISPR8 were excluded from this analysis due to a repeat sequence polymorphism (see text). Read numbers analyzed (n) are indicated. B) The length of the Cmr complex crRNAs is independent of the length of the genome-encoded guide sequence. Deep sequence profiles of crRNAs 4.01 – 4.05 in total P. furiosus RNA (upper panel) and immunopurified Cmr complexes (lower) are shown relative to repeat (black) and guide (green) sequence elements (see also Figure 2A). Dashed lines mark the 3’ ends of the 45-nucleotide crRNA species. (The 5’ end and 3’ end of the 39-nucleotide species are also indicated for crRNA 4.01.) The sequence at the 3’ guide-repeat junction is shown below for crRNAs 4.01, 4.02 and 4.05. Guide sequences are shown in green, and repeat sequences are shown in black. Arrowheads indicate the locations of the 3’ ends of the 45-nucleotide crRNA species.

Figure 4. Antisense RNA found in P. furiosus is cleaved by the Cmr-crRNA complex

A) Antisense RNA from the region of crRNA 1.01 is detected by Northern analysis, and two species are associated with the Cmr complex. Northern analysis of total P. furiosus RNA (T) and RNAs isolated from immune (I) or pre-immune (PI) Cmr2 immunopurifications was probed for antisense (left panel) or sense 1.01 crRNAs (right panel). Colored dots indicate the primary species of antisense RNAs (blue dots) and crRNAs (red dots) specifically associated with the Cmr complex (and correspond with dots in panel B). Sizes of radiolabeled RNA marker (M) are noted. B) The two antisense RNAs associated with the Cmr complex map downstream of a potential fortuitous promoter and correspond to the expected products of crRNA 1.01-guided cleavage. Deep sequencing profiles of Cmr-associated crRNAs in the region encoding crRNA 1.01 are shown. Red indicates RNAs transcribed from the leader region, and blue corresponds to reads from the opposite strand. The positions of CRISPR repeats (black), guide sequences (green) and BRE/TATA promoters (red and blue) are indicated. A blue line represents the apparent full-length antisense RNA observed by deep sequencing total RNA (see Figure S1). Cleavage of the antisense RNA by the Cmr complex 14 nucleotides from the 3’ ends of the 45- and 39-nt species of crRNA 1.01 (red dots) would produce antisense RNA products of the sizes observed by deep sequencing and Northern analysis (blue dots). C) Immunopurified Cmr complexes cleave the antisense RNA. Radiolabeled RNA targets for the antisense RNAs (left panel) and crRNAs (right panel) were incubated in the absence (−) of proteins or in the presence of immune (I) or pre-immune (PI) immunourified preparations, and the products were analyzed following denaturing gel electrophoresis. (The target for the crRNAs is 140 nucleotides in length and corresponds to the full-length antisense RNA (see panel C).) The cleavage products obtained by incubation of the crRNA target with the immunopurified Cmr complex (asterisks) are approximately the same sizes as the endogenous antisense RNA species that are immunopurified with the Cmr complexes (A, left panel). Non-contiguous lanes from the same gel are indicated by dashed lines.

Figure 5. The 5’ repeat tag is required for formation of functional Cmr complexes

A) The 5’ repeat tag is required for Cmr complex function. Reconstitutions were performed with recombinant Cmr1-6 proteins and the 45-nt. wildtype 7.01 crRNA (AUUGAAAG tag) or crRNA 7.01 lacking the tag (−), and activity was tested against the radiolabeled 7.01 target RNA. RNA marker sizes are indicated. The cleavage product observed with the wildtype crRNA is indicated with an asterisk. B) Mutations to the tag sequence prevent function. Reconstitutions were performed with the 45-nucleotide wildtype 7.01 crRNA (AUUGAAAG tag) or crRNA 7.01 with the indicated mutations in the tag sequence (mutated or inserted nucleotides are highlighted in red) and tested in cleavage assays as described in A. The cleavage product observed with the wildtype crRNA is indicated with an asterisk. C) crRNAs with variant tag sequences are underrepresented in the Cmr complex. Deep sequencing profiles of Cmr-associated crRNAs in the region encoding crRNAs 8.08 – 8.11 are shown (see also Figure 2A). The positions of CRISPR repeats (black) and guide sequences (green) are indicated. The 5’ tag sequence is shown below for crRNAs 8.08 - 8.11. An additional nucleotide present in the wildtype tag of crRNA 8.11 is highlighted in red D) Complementarity to the tag does not prevent function. Cmr complexes reconstituted with wildtype crRNA 7.01 were tested for the ability to cleave the 7.01 target RNA or a target with complementarity to the tag sequence (as illustrated). Cleavage products are indicated with asterisks. Non-contiguous lanes from the same gel are indicated with a dashed line.

Figure 6. Engineered crRNAs direct the Cmr complex to cleave intended target RNAs

A) Specific cleavage of novel target RNAs. Cmr complexes were reconstituted with crRNAs designed to target a random sequence (X crRNA, X’ target, orange) or a fragment from the 5’ end of the β-lactamase (bla) mRNA (5’ bla crRNA, bla target, red), and tested for the ability to cleave both target RNAs. Cleavage products are indicated with asterisks. Diagrams illustrate engineered crRNAs with 5’ repeat tag (black) and guide sequence elements (orange or red) interacting with and cleaving the intended 5’ radiolabeled target. B) Directed cleavage of predicted sites in β-lactamase mRNA. Engineered crRNAs target two locations on the full-length bla mRNA. The full length bla mRNA was subject to cleavage by Cmr complexes reconstituted with crRNAs targeting a region near the 5’ end of the mRNA (5’ bla crRNA, red) or a sequence approximately 200 nucleotides from the 5’ end of the mRNA (internal bla crRNA, blue). Cleavage products are indicated by asterisks. Diagrams illustrate engineered crRNAs interacting with and cleaving the bla mRNA.