Fig. 1: A self-selecting CRISPRa piggyBac vector for the rapid generation of stable, high-efficiency CRISPRa cell populations.

a Vector format and selective strategy for the evaluated piggyBac CRISPRa expression-reporter vectors. Expression of the MCP-P65-HSF1 (MPH) activator and dCas9-VP64 is driven by a constitutive, human EF1α promoter. A human H1 promoter drives constitutive expression of a sgRNA complementary to the self-activating (SA) promoter upstream of a GFP reporter. Expression of a Puro^r gene is driven either by its own constitutive promoter (dual promoter), transcriptionally linked to the MPH/dCas9-VP64 (single transcript) or under control of the CRISPRa dependent SA promoter (CRISPRa selection). Gray triangles indicate the location of LoxP sites. PiggyBac engineered K562 populations were generated in triplicate (n = 3 biologically independent samples) for each vector format and enriched with Puromycin selection. sgRNAs complementary to the promoter proximal region of the indicated genes were cloned into a lentiviral vector context containing a mTagBFP2/Zeocin selection cassette (Supplementary Fig. 1b). Following transduction and Zeocin selection target gene expression was evaluated by quantitative RT-PCR (qRT-PCR) (b), and flow cytometry at day 14 post-infection (c, d). Representative histograms for each condition are overlaid with histograms from stained cell populations expressing a non-targeting control sgRNA (c) (gray). Infections were performed in duplicate (n = 2 technical replicates per biological replicate) and averaged. (Median fluorescence intensity (MFI) was normalized to MFI of an antibody-stained sample expressing a non-targeting sgRNA (d, top). Percentage of cells positive by antibody staining is presented (d, bottom) and background staining from a control sample expressing a non-targeting sgRNA is indicated with a dashed horizontal line for each gene. Data are presented as mean values +/− SD. Statistical comparison was performed by an unpaired one-way ANOVA adjusted for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EF1α -Elongation factor alpha, GFP-green fluorescent protein, dCas9-vp64-nuclease dead spCas9 + vp64 activator fusion, P2A-porcine teschovirus-1 2A self-cleaving peptide, HSF-heat shock factor, PD-L1-Programmed death-ligand1 (CD274), CD14-cluster of differentiation 14, CD2-Cluster of differentiation 2, Prom1-prominin-1 (CD133), CXCR4-C-X-C chemokine receptor type 4 (CD184). Source data are provided as a Source Data file.