DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Cas9 protein expression, which persisted after G1 arrest (Fig. 1B). Sequences corresponding to several genomic sites were cloned into a altered pKLV-gRNA plasmid that contained a guide RNA (gRNA) expression cassette and that harbored the Thy1.1 cell surface marker. Nucleofection of the gRNA plasmid into G1-arrested cells resulted in 90% Thy1.1 positivity (Fig. 1C). G1-arrested cells treated with doxycycline and transfected using nucleofection (nucleofected) with a gRNA expression vector designed to target the endogenous enhancer of (gtarget site (Fig. 1D). To ensure that Cas9-generated DSBs elicit a canonical DNA damage response (DDR) in our system, we mapped -H2AX formation in cells nucleofected with gor no gRNA as a control. Consistent with the findings of prior studies, the -H2AX modification extended for several hundred kilobases on either side of the break site in cells nucleofected with g(Fig. 1E). No -H2AX domain name was observed in the control cells or at other loci in glocus, which is usually constitutively expressed in our cell collection. G1-arrested, cells were nucleofected with a gRNA targeting a site within intron 6, about 6.3 kb downstream of the promoter (Fig. 2A). As expected, we observed high levels of prolonged DSBs at the target site at 24?h postnucleofection (Fig. 2B). A prolonged break within the gene body significantly reduced the levels of its corresponding mRNA, as measured by reverse transcription (RT)-quantitative PCR (qPCR) analysis at 24?h postnucleofection (Fig. 2C). In contrast, expression of a control gene, promoter (Fig. 2A and ?andE).E). As observed for breaks within the gene body, a DSB upstream of the same transcriptional unit led to a nearly identical reduction in total and nascent transcripts (Fig. 2F and ?andG).G). We conclude that single DSBs in G1 phase can silence expression of proximal genes, even when they do MG149 not directly interrupt the transcriptional unit. Open in a separate windows FIG 2 Gene body or 5 DSBs attenuate expression of the endogenous gene. (A) Schematic of the locus. gRNA target sites are denoted by yellow arrows. The qPCR primers used to detect transcripts MG149 are shown as reddish arrows. The distances between gRNA target sites and the promoter are indicated. chr13, chromosome 13. (B) Schematic of the Southern blotting strategy for detecting cleaved alleles at the intronic gRNA target site (gintron) (top) and Southern MG149 blot CIT showing intact and slice alleles 24?h after nucleofection of doxycycline-treated, G1-arrested cells with the vacant gRNA vector or gintron (bottom). (C) RT-qPCR analysis of total transcript levels (primer pairs P1 and P2) and a control gene, intron MG149 6 (gintron). The MG149 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 explained in the story to panel B, for gRNA targeting the region 9.5?kb upstream of the promoter (g5). (F) RT-qPCR analysis of total transcript levels, as explained in the story to panel C, for cells nucleofected with a gRNA targeting.
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