Figure 2.

Design and delivery of multi-gRNA PB vectors for CRISPRi and CRISPRa targeting of the TCF4 gene. (a) Representative western blot of TCF4 protein expression in hPSCs. Letters a and b denote the two observed bands. (b) Top, Schematic of the TCF4 gene exons containing potential TSS locations for the putative protein isoforms shown in (a). Numbers below exons correspond to the primer pairs used for RT-qPCR. Exon locations are modified from Sepp et al.²⁷, with approximate predicted molecular weights of resulting protein isoform(s) listed above each starting exon in italics, with tested exons highlighted in red. The asterisks under exon 3d indicates there was no detectable PCR signal for this exon with two separate primer pairs (data not shown). Bottom, Exon-specific expression of TCF4 transcripts from RT-qPCR of two independent H1 hPSC samples and normalized to GAPDH. -RT lane denotes the no reverse transcriptase control cDNA reaction. Data is shown as mean +/− s.e.m. of two independent biological samples. (c) Overview of multi-gRNA PB vector cloning, delivery, and selection. (d) Representative images of mRFP fluorescence in dCas9-KRAB-PB clones K1 and K2 (top panels) and dCas9-VPR-PB clones V1 and V2 (bottom panels). Cells are counterstained with DAPI (blue). Scale bar = 100 μm. (e) PB vector copy number in dCas9-KRAB and dCas9-VPR clones as determined by ddPCR quantification of mRFP gene. Data is shown as the mean of three experiments with error bars as +/− s.e.m.