Fig. 2: Repression versus cutting of endogenous genes using tgCRISPRi and CRISPR in Drosophila.
From: tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9
a Graph shows comparison of body color phenotypes in F1 and F2 progeny generated by crossing F1 males carrying the vasa-Cas9 source and either tgRNAs or full-length gRNAs to females homozygous for reference y or e mutations (n = 5 individual crosses). Data were analyzed using One-way ANOVA. Data plotted as mean ± standard deviation, ****p < 0.0001. b Graph shows comparison of NHEJ events generated by either tgRNA or gRNA in F1 progeny. Data were generated using the ICE tool (n = 5 × 12 flies per target gene). c Comparison of CRISPR and tgCRISPRi DNA sequence Chromatograms reveals differential generation of NHEJ events at y gene target sites. The control y + DNA sequence around the tg/gRNA-y2 and tg/gRNA-y1 target sites is shown on top of the 1st and 4th row, respectively. Chromatograms for control (+/vasa-Cas9) animals (1st row) show no NHEJ events revealed as double peaks. Upon introduction of tgRNA-y2 and y1, peaks appear similar to the control (2nd row). In gRNA-y2 or y1 expressing animals (3rd row), however, multiple peaks appear around the target sites, confirming double-strand cleavage and imprecise DNA repair. d Detection of tgCRISPRi and CRISPR-induced mutations for guides targeting the y gene. The percentage of indels was quantified using the Synthego ICE online tool. F1 progeny from control, tgRNAs, and gRNAs master flies were sequenced. For controls and tgRNAs, 100% of F1 alleles were wild type. For full-length gRNAs, however, 100% of F1 alleles were NHEJ events. Sequences on the right side show the mutations with the sizes of deletions (−) and insertions (+), with the percentage of indels and KO score for each allele. e RT-qPCR of e gene from control (Ct = +/vasa-Cas9) and tgRNA-e(1 + 2) (tgRNA/vasa-Cas9) adult flies. Transcript levels of rp49 were used as a reference. Error bars represent standard deviation of the mean of three independent experiments and P values derived using a two-tailed unpaired T-test, ****p < 0.0001.
