Fig. 1: Specific and durable transcriptional silencing by CRISPRoff in primary human T cells.
From: Integrated epigenetic and genetic programming of primary human T cells
a, Comparison of KD efficiency of CD151 across seven CRISPRoff mRNA designs over a series of mRNA doses. CD151 expression was assessed using flow cytometry 5 days after electroporation. We modeled CD151 positivity as a function of dose and mRNA variant and then computed a P value for the difference between CRISPRoff 7 and the rest of the mRNA variants using the standard error (Methods). P values were then adjusted using the Benjamini–Hochberg procedure. CRISPRoff 7 was the most potent CRISPRoff mRNA variant as assessed by the degree of CD151 silencing across CRISPRoff doses (n = 2 donors; CRISPRoff 1, ***P = 0.00022; CRISPRoff 2, **P = 0.011; CRISPRoff 3, **P = 0.0096; CRISPRoff 4, **P = 0.001; CRISPRoff 5, **P = 0.0044; CRISPRoff 6, *P = 0.04). b, Comparison of Cas9 (black), CRISPRi (red) or CRISPRoff (blue) mRNA KO or KD activity on CD151, CD55 and CD81 loci over a time course of 28 days after electroporation. Black arrows along the x axis indicate restimulations with anti-CD2/CD3/CD28 soluble antibodies (day 9, day 18 and day 27 after electroporation) (n = 4 donors except on day 10, where n = 2 donors; mean ± s.d.). c, Representative flow cytometry histogram plots of CD151 KD (or KO) by CRISPRoff, CRISPRi or Cas9 on day 5 and day 28 after electroporation. d,e, Transcriptomic assessment by RNA-seq of CRISPRoff activity and specificity upon silencing of CD55 (d) or CD81 (e) relative to NTC. Cells were electroporated with CRISPRoff mRNA and an sgRNA targeting CD55 or CD81 or NTC. Cells were harvested 28 days after electroporation for RNA extraction. Yellow dots indicate significantly downregulated DEGs and gray dots have no significance (empirical Bayes moderated statistics with Benjamini–Hochberg FDR control, adjusted P < 0.05; CD55 adjusted P = 2.01 × 10−5 and CD81 adjusted P = 1.65 × 10−4; n = 2 donors). f, Comparison of CpG methylation analyzed by WGBS within a 20-kb window centered on the CD55 TSS. CGIs are depicted in green and the sgRNA targeting site is annotated. Tracks represent samples electroporated with CRISPRoff mRNA and an sgRNA targeting the CD55 TSS or NTC for two independent donor replicates (D1 and D2). Cells were collected at 30 days after electroporation. g, The Manhattan plot displays DMRs between cells treated with CRISPRoff and an sgRNA targeting CD55 or NTC and analyzed by WGBS (cells were collected at 30 days after electroporation). Red dots represent DMRs that gained DNA methylation in the targeting sgRNA samples. Blue dots represent DMRs that gained DNA methylation in NTC samples. The arrow denotes the genomic position of CD55 (n = 2 donors performed in technical replicates). h, Day 27 transcript levels of FAS, PTPN2, RC3H1 (Roquin 1) and SUV39H1 relative to NTC as measured by RT–qPCR (n = 3 donors). i, Transcriptomic assessment by RNA-seq of CRISPRoff activity upon silencing of FAS relative to NTC. Cells were electroporated with CRISPRoff mRNA and an sgRNA targeting FAS or an NTC and then harvested at 7 days after electroporation for RNA extraction. The yellow dot indicates the target gene, which is significantly downregulated, and gray dots have no significance (empirical Bayes moderated statistics with Benjamini–Hochberg FDR control, adjusted P < 0.05; FAS adjusted P = 3.35 × 10−20; n = 4 donors).
