Panel A shows Sanger sequencing chromatograms of genomic DNA at the CRISPR-Cas9 editing site, comparing control, WDR75 wild type, and WDR75 T46I (M1), with sequence peaks clearly visible. Panel B shows alignments of sequencing data from TOPO-TA cloned RT-PCR products of edited U2OS cells. The reference and sample sequences for WDR75 wild type and WDR75 T46I (M1) are compared, with mismatched bases highlighted in red. Panel C shows Northern blot analysis of U2OS cells expressing empty vector, WDR75 wild type, or WDR75 T46I (M1). Different probes detect pre-rRNA intermediates and mature rRNAs, with bands corresponding to multiple RNA species. Adjacent graphs present ratios of these RNA forms, with significant differences marked by asterisks.
U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site (guide sequence) of CRISPR/Cas9 modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData FS5.