Fig 1.
Reconstitution and RNA cleavage activity of a T. thermophilus Csm complex (TthCsm) purified from E. coli with a defined crRNA species.
(A) Components of the CRISPR locus and effector complexes of the T. thermophilus Type III-A Csm system. The complex is shown with 5 copies of Csm3 and 4 copies of Csm2, but complexes with different numbers of these two subunits also exist. The CRISPR-4 locus associated with the system is shown (repeat is designated by R and spacer by S). The spacer 4.5 used for complex reconstitution encodes for one of the most abundant crRNAs found in the host organism [21]. (B) Reconstitution and purification of TthCsm in E. coli. A plasmid containing genes encoding for Cas10/Csm1, and Csm2-5, with a His10 tag on Csm5, was co-transformed into E. coli with a plasmid containing genes for expression of T. thermophilus Cas6A and a single CRISPR array containing one copy of spacer 4.5. The purification steps are indicated. (C) TthCsm was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis following purification. Csm subunits are labeled, and a molecular weight ladder (M) is in the left lane (masses are given in kilodaltons). A GroEL contaminant (asterisk) was also identified by mass spectrometry (S2 Table). (D) TthCsm-mediated cleavage of a complementary (C) or noncomplementary (NC) 32P-labeled ssRNA oligonucleotide was tested in the presence of 2 mM MgCl2. Samples taken at 0, 5, 30, and 60 minutes after TthCsm addition were analyzed by denaturing PAGE. (E) Schematic of crRNA processing in Type III CRISPR-Cas systems is shown on the left. Pre-crRNAs are cleaved by Cas6 to generate an intermediate, which is then trimmed at the 3’-end, resulting in mature crRNAs. On the right, nucleic acids associated with the Csm complex were extracted and analyzed by denaturing PAGE. An ssDNA oligonucleotide ladder (M) was loaded in the right-most lane and nucleotide lengths are indicated.
Fig 2.
TthCsm specifically recognizes and binds ssRNA through complementarity to its crRNA.
(A) An increasing concentration of TthCsm, from 0–300 nM, was incubated with 0.5 nM 32P-labeled ssRNA target that was complementary (C) or noncomplementary (NC) to the crRNA guide sequence, and analyzed by an EMSA. (B) 100 nM TthCsm was incubated with 0.5 nM 32P-labeled target ssRNA and increasing concentrations of unlabeled complementary ssRNA (ssRNA, C), noncomplementary ssRNA (ssRNA, NC), complementary ssDNA (ssDNA, C), or noncomplementary ssDNA (ssDNA, NC) competitor (0–1 μM). Samples were assayed for binding of the probe using an EMSA, as in (A).
Fig 3.
TthCsm binds and cleaves its ssRNA target optimally at high temperatures.
(A) TthCsm was pre-incubated in the absence of metal ions with complementary ssRNA at either 65°C or 37°C (pre-incubation), and then cleavage was initiated by addition of MgCl2 and continued at either 65°C or 37°C, as indicated (assay). Reactions with (+) or without (-) TthCsm added are shown. (B) TthCsm was incubated with a 32P-labeled complementary ssRNA target at 65°C or 37°C, and binding was measured by an EMSA, as in Fig 2A. TthCsm was added to a concentration of 0–1200 nM at 37°C or 0–300 nM at 65°C.
Fig 4.
RNA-guided ssDNA cleavage by TthCsm.
(A) Schematic of the DNA cleavage reaction. TthCsm was mixed with 5′-32P-labeled ssDNA oligonucleotide and an unlabeled complementary ssRNA oligonucleotide in the presence of 1 mM EDTA. The reaction was initiated by the addition of 5 mM MnCl2. (B) TthCsm-mediated ssDNA cleavage was tested in the presence of complementary (C) or noncomplementary (NC) ssRNA, as described in (A). Time points were taken at 0, 10, 15, and 30 min, and cleavage products were analyzed by denaturing PAGE. (C) As in (B), but using unlabeled ssRNA substrates that are complementary to the crRNA guide sequence, but have an 8 nt-long 3′ flanking region that is either noncomplementary (3′-nc), complementary (3′-c), truncated (Δ3′), or both extended to 20 nt long and noncomplementary (3′-ext).
Fig 5.
The HD domain is required for ssDNA cleavage by TthCsm.
(A) Schematic of TthCsm, and domain architecture of the T. thermophilus Csm1 component protein are shown. The HD and palm polymerase domain are indicated, and the approximate positions of the HD and GGDD mutations tested in (B) and (C) are shown. N and C termini of Csm1 are indicated. (B) ssDNA cleavage mediated by the wild-type TthCsm (WT), TthCsm containing an HD domain mutation (H18A/D19A, indicated by HDm), and TthCsm complex containing a palm polymerase domain mutation (G630A/G631A/D632A/D633A, indicated by GGDDm) was tested as in Fig 4B. Time points were taken at 0, 5, 10, 15, and 30 min and analyzed by denaturing PAGE. (C) WT, HDm or GGDDm TthCsm complexes were tested for ssRNA cleavage, using same conditions as in (B), but with only complementary, radiolabeled ssRNA added.
Fig 6.
TthCsm-mediated ssDNA cleavage is sequence-independent and endonucleolytic.
(A) TthCsm-mediated cleavage of a 5′-32P-radiolabeled complementary or noncomplementary dsDNA (dsDNA, C or NC), ssDNA (C or NC), or a duplex with a 40-nt long mismatch in the center (bubble DNA) was tested in the presence of complementary target ssRNA, as in Fig 5B. (B) TthCsm was incubated with 5′-32P-radiolabeled 25 nt-long ssDNA substrates with different sequences in the presence of a complementary target ssRNA and MnCl2. Sequences included all four nucleotides (all nt), all except adenines (-A), thymines (-T), guanines (-G), or cytosines (-C). The reaction was carried out for 60 minutes, either with (+) or without (-) TthCsm added.
Fig 7.
TthCsm-mediated DNA cleavage does not require RNA cleavage.
(A) TthCsm-mediated cleavage of a complementary ssRNA either with (deoxy-RNA) or without (RNA) deoxynucleotides adjacent to the regularly spaced cleavage sites was tested and analyzed by denaturing PAGE, as in Fig 5C. (B) ssDNA cleavage by TthCsm was assayed as in Fig 5B, but with the RNA substrates in (A). (C) The wild-type TthCsm (WT) or TthCsm containing a D34A or D34N mutation in the Csm3 subunit (D34A, D34N) were tested for cleavage of a complementary ssRNA target, as in (A). (D) WT, D34A, or D34N TthCsm complexes were tested for ssDNA cleavage as in (B).
Fig 8.
A conserved mechanism for co-transcriptional DNA and RNA targeting by Type III-A CRISPR-Cas effector complexes.
During transcription, RNA polymerase transiently unwinds double-stranded DNA as it synthesizes a messenger RNA transcript (mRNA) that is complementary to the template strand. Transcription across a region of the genome that is complementary to the crRNA leads to the production of an mRNA containing a region complementary to the crRNA in the Csm complex. The Csm complex would recognize and bind the mRNA through base-pairing interactions with its crRNA, leading to activation of the sequence-independent DNA endonuclease activity of the HD domain in Csm1. This would lead to Csm-mediated cleavage of transiently unwound ssDNA, while Csm is tethered via the RNA. Self-targeting is avoided by preventing cleavage when the 3′ flanking region of the RNA is complementary to the 5′ crRNA tag. Following DNA and RNA cleavage, the Csm complex dissociates from its targets.