Fig. 1: TERRA depletion by the RCas9 system reduces alternative lengthening of telomeres.

a RCas9-sgRNA system to deplete TERRA RNA. The RCas9 system contains dCas9 fused to PIN RNA endonuclease. b Northern blot analysis showing TERRA levels in TERRA knockdown U2OS cells (sgTER_1C6, sgTER_1C21) or control cells (sgSense, sgλ2). c Quantification of TERRA intensity in (d). P-values, two-sided Mann-Whitney U-test. Bars, mean ± SEM. n, cell number. d Immuno-RNA FISH to detect TERRA RNA, TRF2, and PML in U2OS cells expressing RCas9-sgRNAs. Arrowheads indicate APBs containing big TRF2 foci colocalized with PML. e Quantification of top 5% TRF2 foci intensity in (d) using 3D images by Imaris software. Bars, median. n = number of TRF2 foci. Data of three independent experiments. P-values, two-sided Mann-Whitney U test. f Quantification of APB foci in TERRA knockdown cells (sgTER_1C6, sgTER_1C21) or control cells (sgSense, sgλ2). APB foci were determined by the extensive TRF2 staining (> top 5% of TRF2 foci intensity in the control cells) with PML staining. Bars, mean ± SEM. n, cell number. Data of three independent experiments. P-values, two-sided Mann-Whitney U-test. Mean values of each group shown on the top of figures. g Quantification of telomeric R-loops by qPCR after DNA-RNA immunoprecipitation using S9.6 antibody. P-values, two-tailed student’s t-test. bars, mean ± SEM. h Telomere length measured by TRF analysis in cells expressing RCas9-sgRNAs at PD6 after selection. The same amount of DNA as TRF assay was loaded in the EtBr gel. The mean of telomere length was quantified (top-right). P-values by two-tailed Student’s t-test. Bars, mean ± SEM. Three independent experiments were averaged. c, e, g Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file.