Fig. 3: Durable multiplexed gene silencing.
From: Integrated epigenetic and genetic programming of primary human T cells
a, Graph showing the number of live T cells following cell editing by CRISPRoff and Cas9 when targeting either three, four or five genes simultaneously as compared to an empty electroporation (EE) control. Live-cell counts were measured 5 days after electroporation (n = 4 donors; mean ± s.e.m.; two-sided Welch’s t-test: Cas9, three genes, **P = 0.004; Cas9, four genes, ***P = 0.00015; Cas9, five genes, ***P = 0.00013; NS, not significant). b, Plot comparing CRISPRoff versus Cas9 multiplexed gene silencing efficiency targeting three genes (CD151, CD81 and CD44), four genes (CD151, CD81, CD44 and CD55) or five genes (CD151, CD81, CD44, CD55 and CD46) at 5 days and 30 days after electroporation. The percentage of cells with all genes silenced was calculated from flow cytometry analysis (n = 4 donors; mean ± s.e.m.). c, A representative flow plot of cells targeted for triple-gene (CD151, CD81 and CD44) silencing (top) or NTC (bottom). Cells were analyzed at 30 days after electroporation. Top, cells were first gated on CD44-silenced cells and the represented population shows CD81 and CD151 silencing. d, An RNA-seq log2 CPM (normalized counts per million) plot showing triple-target-gene KD in cells electroporated with CRISPRoff mRNA and sgRNAs targeting FAS, RC3H1 and SUV39H1 or an NTC sgRNA. Cells were collected for RNA-seq 7 days after electroporation (n = 4 donors; mean ± s.e.m.). e, Pie charts depicting the outcomes of three-gene, four-gene or five-gene silencing or KO shown in b. Data are representative of one donor.
