Fig. 2. RTCB ligase repairs RNA cleaved by the type III CRISPR complex.
(A) Human RNA ligase RTCB joins RNA ends that are produced in tRNA splicing (top) and non-canonical XBP1 mRNA splicing (bottom) during the unfolded protein response. We hypothesized that the ligase activity of RTCB is involved in the RNA repair of CRISPR-guided RNA breaks (middle). (B) Western blot with anti-RTCB or anti-ACTB (loading control) antibodies was performed with lysates of 293T cells with (+) or without (−) RTCB depletion. See the uncropped images in fig. S3. (C) The PARK7 transcript was targeted with SthCsm (guide 2) in 293T cells with (+) or without (−) RTCB depletion. The PARK7 transcript was quantified with RT-qPCR and normalized to ACTB and non-targeting guide RNA. Data are shown as the mean ± standard deviation of three biological replicates. *P < 0.05, ***P < 0.001; one-way analysis of variance (ANOVA) with Tukey HSD post-hoc comparisons. (D) qPCR products in (C) were sequenced, and resulting reads were aligned to the reference sequence of the PARK7 transcript (NM_007262, GenBank). Graphs show sequencing depth (y-axes) at the amplified region of the transcript (x-axes). Every line shows a biological replicate (n = 3). The horizontal black bar indicates a region complementary to the guide RNA of the SthCsm complex. Vertical dotted lines mark predicted positions of RNA breaks. (E) Quantification of deletions in the target region of the PARK7 transcript. Data is shown as the mean ± standard deviation of three biological replicates. Welch’s t-test was used to compare mean values. *** p < 0.001. (F) RTCB deficient cells were transfected with a plasmid expressing RTCB (pCMV-RTCB), and the efficiency of CRISPR RNA-guided programmable deletions in PARK7 was quantified as in panels C-E. * p < 0.05; OneWelch’s t-test. (G) Immunostaining of cells expressing Flag-tagged Csm complex with (NLS-Csm) or without (Csm) NLS-tag. Scale bars are 10 μm. (H-J) Knockdown of PARK7 and XIST transcripts with cytoplasmic SthCsm (no NLS) (H, I) and knockdown of XIST with nuclear Csm (NLS-tagged) (J) was quantified with RT-qPCR and normalized to ACTB and non-targeting control. “wt” – nuclease-active SthCsm, “dead”– catalytically inactivated SthCsm (Csm3D33A mutation). *P < 0.05, ns – non-significant; Welch’s t-test. Data is shown as mean ± SD (n = 3).