Figure 2: Deletion of the 20q-TERRA locus in different human cell lines using the CRISPR-Cas9 system.

(a) Snapshot depicting, from top to bottom, putative promoter regions (Pr1, Pr2, Pr2 and Pr4), gRNA position, genomic coordinates, RNA-seq from TERRA IP and input¹³, and RNA polymerase 2A (POL2A) and CTCF ChIP-seq data. S2, E1, S2 and E2 are the names of the different gRNAs. (b) Graph shows the relative fold increase in firefly luciferase activity seen in the pGL3-containing promoter regions (Pr1-4) relative to the empty vector after normalization to renilla activity. (Mean values±s.e.m., n=3 independent experiments). p21 promoter serves as positive control. One-way Anova with the Dunnett’s post test was used for statistical analysis (*P<0.05 and ***P<0.001). (c) Representative image of the HeLa clones homozygous for the 20q-TERRA deletion during expansion. Zoom areas are shown. Scale bar, 500 μm and (zoom) 250 μm. (d) Percentage of viability of the different homozygous clones for the 20q and Xp deletion on expansion of the three different cell lines. (e) Ethidium bromide gels showing the CRISPR allele for the deletion of the 20q and Xp loci detected by PCR in different clones of the U2OS cells. The white strips in the gels indicate the removal of irrelevant samples from that gel. (f) Schematic of the sequencing of the CRISPR allele for the deletion of the 20q (clones A2, B4 and C4) and Xp (clone D8) loci compared with the WT allele. Slashes (//) represent omitted DNA sequence. The size of the omitted sequence is shown. Dashes (-) represent the deleted sequence. The size of the deleted sequence is shown. gRNAs are shown in blue.