Fig. 2

Confirmation of the CCAT1 knockouts and qPCR analysis. a CCAT1 wild-type locus and its knockout alleles by “CRISPR excision”, “CRISPR du-HITI”, and “CRISPR HDR”. Location of sgRNA binding sites (sgRNA b. s.), primers to perform PCR analysis (Table 4), and inserted fragments are shown. “CRISPR excision” of exon I (Ex. I) is confirmed by a 409 bp PCR fragment using two primers at either side of exon I. “CRISPR du-HITI” is confirmed by a PCR product of 495 bp using a primer in the insert and the other in the flanking region. The insert used for “CRISPR du-HITI” could express EGFP (bright green) or puroR (light blue) under CAG promoter. In this strategy when two alleles are targetted simultaneously, then the cells show both green fluorescence and resistance to puromycin dihydrochloride. “CRISPR HDR” was performed on exon II using an insert containing DSred (red in color), CMV, puroR, IRES, GFP, PA (poly adenylation signal, purple in color). In “CRISPR HDR” strategy cells with only one targetted allele could show both green fluorescence and resistance to puromycin dihydrochloride. PCR analysis using a primer in the insert and the other in the flanking region results in a 1627 bp product (Table 4). b Gel electrophoresis of PCR fragments for wild-type (w.t.) (a), CRISPR excised (b; clone # 20), “CRISPR du-HITI” knockout (c; clone # 27), and “CRISPR HDR” alleles (e; clone # 2i) according to the maps in panel A. Lane d is DNA size marker. c CCAT1 transcript relative to β-actin levels in wild-type and “CRISPR excision” knockout HT-29 colon adenocarcinoma cells. d CCAT1 transcript relative to β-actin levels in wild-type and “CRISPR HDR” knockout HCT-116 colon adenocarcinoma cells. e CCAT1 transcript relative to β-actin levels in wild-type and “CRISPR du-HITI” knockout SW-480 colon adenocarcinoma cells. In panels, C, D, and E the absolute copy number for CCAT1 and β-actin transcripts were quantified based on the related standard curves, and for three series of cell line cDNAs, the quantity of the CCAT1 transcript divided by the quantity of the β-actin was plotted. The statistical differences between the wild-type and knockout cell lines are analyzed by Mann-Whitney U test