FIG 2.

Gene body or 5′ DSBs attenuate expression of the endogenous Irf4 gene. (A) Schematic of the Irf4 locus. gRNA target sites are denoted by yellow arrows. The qPCR primers used to detect Irf4 transcripts are shown as red arrows. The distances between gRNA target sites and the Irf4 promoter are indicated. chr13, chromosome 13. (B) Schematic of the Southern blotting strategy for detecting cleaved alleles at the Irf4 intronic gRNA target site (gIrf4 intron) (top) and Southern blot showing intact and cut Irf4 alleles 24 h after nucleofection of doxycycline-treated, G₁-arrested LigIV^−/−:iCas9 cells with the empty gRNA vector or gIrf4 intron (bottom). (C) RT-qPCR analysis of total Irf4 transcript levels (primer pairs P1 and P2) and a control gene, Tnks2, at 24 h after nucleofection with a gRNA targeting Irf4 intron 6 (gIrf4 intron). The transcript levels relative to those in cells nucleofected with an empty gRNA control vector (gEmpty) are shown. (D) RT-qPCR analysis of nascent transcript levels from samples for which the results are shown in panel C, performed using the Click-iT nascent RNA capture technology. Cells were pulsed with 5-ethynyl uridine (EU) 1 h prior to harvesting for RNA isolation. (E) Southern blot schematic and Southern blot, as described in the legend to panel B, for gRNA targeting the region 9.5 kb upstream of the Irf4 promoter (gIrf4 5′). (F) RT-qPCR analysis of total transcript levels, as described in the legend to panel C, for cells nucleofected with a gRNA targeting the region 9.5 kb upstream of the Irf4 promoter (gIrf4 5′). (G) RT-qPCR analysis of nascent transcript levels, as described in the legend to panel D, from samples for which the results are shown in panel F. Data from panels C and D and from panels F and G represent those from 3 independent experiments (n = 3). Error bars show the SEM. *, P < 0.05; **, P < 0.01; n.s., not significant.