Fig. 2: CRISPRoff silencing at genes that lack CGI annotations.
From: Integrated epigenetic and genetic programming of primary human T cells
a, Comparison of Cas9 KO (black) or CRISPRoff KD activity with either a single sgRNA (light blue) or a pool of three sgRNAs (dark blue) at CD5 over a time course of 30 days after electroporation (n = 2 donors). b, Representative flow cytometry histogram plots of CD5 KD (blue) or KO (black) compared to NTC (gray) at 30 days after electroporation. c, Comparison of Cas9 KO (black) or CRISPRoff KD activity with either a single sgRNA (light purple) or a pool of three sgRNAs (dark purple) at LAG3 over a time course of 31 days after electroporation (n = 2 donors). d, Representative flow cytometry histogram plots of LAG3 KD (purple) or KO (black) compared to NTC (gray) at 31 days after electroporation. e, Comparison of Cas9 KO (black) or CRISPRoff KD activity with either a single sgRNA (light green) or a pool of three sgRNAs (dark green) at PDCD1 over a time course of 30 days after electroporation (n = 2 donors). f, Representative flow cytometry histogram plots of PD1 KD (green) or KO (black) compared to NTC (gray) on day 31 after electroporation. g, Comparison of Cas9 KO (black) or CRISPRoff KD activity with either a single sgRNA (light pink) or a pool of three sgRNAs (dark pink) at CD39 over a time course of 35 days after electroporation (n = 2 donors). h, Representative flow cytometry histogram plots of CD39 KD (pink) or KO (black) compared to NTC (gray) at 31 days after electroporation. i, Comparison of Cas9 KO (black), CRISPRi (gold) or CRISPRoff activity with a single sgRNA (orange) targeting CD45 over a time course of 24 days after electroporation (n = 2 donors). j, Representative flow cytometry histogram plots of KO (black), CRISPRi KD (gold) or CRISPRoff KD (orange) compared to NTC (gray) at 24 days after electroporation. In a,c,e,g,i, black arrows along the x axis indicate restimulations with anti-CD2/CD3/CD28 soluble antibodies. k, Fold change in surface protein expression (% positive cells) from the first time point to the last time point for cells treated with CRISPRoff mRNA and the most potent guide targeting the TSS of each respective gene. The number of CpG dinucleotides within ±500 bp of the TSS of each gene is shown on the x axis. Red dots indicate genes with CGI annotations in the UCSC genome browser. Black dots indicate genes that do not have a CGI annotation.
