Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble

Fig. 1. Tethering of TthCsm to a transcription elongation complex (TEC) by a complementary nascent RNA transcript. a Schematic of the TthCsm complex and the TEC. TthCsm subunits are labeled. RNAP; RNA polymerase. The sequence in the RNA transcript that is complementary to the crRNA in TthCsm is highlighted in orange. A 5´-desthiobiotin moiety on the template strand of DNA in the TEC is labeled. b Schematic of the pull-down assay. The TEC was immobilized on streptavidin beads using a desthiobiotin group on the DNA. TthCsm was incubated with the TEC, after which the beads were washed to remove unbound complexes, and biotin was added to elute the TECs from the beads. c Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified T. thermophilus RNAP (α, β´, β, σ, ϖ) and TthCsm (Cas10/Csm1, Csm2-Csm5) complexes. A molecular weight marker was run for comparison in the leftmost lane and sizes are indicated in kilodaltons. d Pull-down assays were done with TECs containing a target (left), non-target (middle), or target RNA transcript added in trans (right). The elutions were analyzed by SDS-PAGE. The presence of RNAP and TthCsm subunits is indicated. The positions of the…

Fig. 2. Visualization of TthCsm tethered at the TEC. Negative-stain 3D EM reconstructions of the TthCsm-TEC complex are shown. TthCsm is colored gray, and the RNAP is colored light blue. The 3D models are numbered from 1-10. The orientation of TthCsm is aligned in all of the 3D models and a colored gradient arrow is used to illustrate how RNAP changes its orientation from in front to behind TthCsm. The boxed model 8, which has the most well-resolved features on the RNAP, was used for structural modeling in Fig. 3

Fig. 3. Cryo-EM structures of target RNA-bound TthCsm and modeling of the TthCsm-TEC complex. a Cryo-EM structure and model of target RNA-bound TthCsm. TthCsm subunits, crRNA, and target RNA are labeled in the different views. Each view is rotated by 90˚, as indicated by arrows. b Detailed view of the discontinuous crRNA and target RNA hybrid, showing the base pairs fit into the EM density of the RNA hybrid. A loop in each Csm3 subunit inserts after every 5-nt segment and flips out the 6th base. The EM density of the crRNA:target RNA hybrid and the modeled crRNA strand are colored dark blue, and the modeled target RNA strand is colored red-orange. The EM density and model corresponding to the third Csm3 subunit (Csm3.3) is colored light blue. c Models of RNA-bound TthCsm and the TEC complex fit into the negative stain EM map. The HD domain of Cas10/Csm1 is colored pink, the nontemplate strand (NTS) and template strand (TS) are colored red, the RNA transcript is colored orange, and the crRNA is colored dark blue. A dotted orange line is used to represent the segment of RNA between the RNA exit channel and the TthCsm bound target sequence. TthCsm is colored gray and the protein subunits of…

Fig. 4. Target preference of TthCsm at the transcription bubble. a Cleavage of nucleic acids by TthCsm in the TthCsm-TEC complex was initiated by addition of MgCl2 or MnCl2 and reactions were incubated for 30 min at 65 ˚C. Products were analyzed by denaturing PAGE and SYBR gold staining. The nontemplate strand (NTS DNA), template strand (TS DNA), RNA transcript (RNA), and crRNAs contained in the TthCsm (Csm crRNAs) were loaded individually on the left half of gel for comparison. b TthCsm-catalyzed cleavage of the 5´-radiolabeled NTS. The substrates tested were the eluted TthCsm-TEC sample from the pull-down (left), the NTS DNA alone with the target RNA transcript, which includes a sequence complementary to the TthCsm crRNA (center), and an R-loop bubble composed of NTS DNA, TS DNA, and the target RNA transcript (right). A ssDNA marker was run in the leftmost lane (M), and the position of the unpaired region is shown. Uncropped gel images for (a, b) are available online in the Source Data file

Fig. 5. Testing co-transcriptional DNA and RNA targeting activities of SepCsm. a Schematic of the TEC used in the co-transcriptional DNA cleavage assay. The TEC was assembled with RNAP, a short RNA primer, nontemplate strand (NTS DNA), and template strand (TS DNA) of DNA. Ribonucleotides (rNTPs) were added to initiate transcription across the DNA and produce an RNA transcript. The NTS DNA and corresponding RNA transcript contains a target sequence (highlighted in orange) that is complementary to the crRNA guide sequence in SepCsm. b Co-transcriptional cleavage of 5´-radiolabeled RNA by SepCsm, analyzed by denaturing PAGE. Transcription was first initiated across the target sequence in the DNA by addition of rNTPs, followed by addition of SepCsm after 10 min. For reactions with both SepCsm and rNTPs added, samples were taken at 30, 60, 90, and 120 min after SepCsm addition. For all other reactions, samples were taken at 120 min. An RNA Decade™ marker (M) is loaded in the leftmost lane. c Co-transcriptional cleavage of 5´-radiolabeled NTS DNA by SepCsm, analyzed by denaturing PAGE. The assay was performed as in (b). The molar ratio of NTS to TS used in TEC assembly is given above the gel.…

Fig. 6. Model for transcription-dependent immunity by RNA-guided Type III CRISPR-Cas systems. Transcription across a DNA target generates a nascent transcript that is complementary to the crRNA of Csm (complementary target sequence in RNA is colored orange). Binding to the transcript flexibly tethers TthCsm to the TEC, but does not lead to an interaction of TthCsm with the RNAP or the DNA in the transcription bubble. Csm cleaves transcripts using its Csm3 subunit, instead of cleaving the ssDNA at the transcription bubble. While bound to RNA, Csm also synthesizes cyclic oligoadenylate (cOA) molecules, which binds to and activates the Csm6 RNase for transcript degradation