Figure 5
From: Effective CRISPR interference of an endogenous gene via a single transgene in mice
Specificity of CRISPRi. (a) Quantitative RT-qPCR analysis of transgene, Tnfsf11, Akap11, and Fam216b mRNA transcripts in kidney from 5-week-old mice of WT (n = 6) and TG (n = 6) mice from line 157. Both sexes were included. Transgenic compared to WT littermates of the same line using Student’s t test. (b) RNA-sequencing normalized expression patterns are plotted for UAMS-32 cells stably expressing dCas9::KRAB and either the sgRNA targeting Tnfsf11 (sgRNA-3) or the exogenous SV40 promoter sequence (sgRNA-SV40). No major transcriptome alterations occurred in the sgRNA-3 expressing cells. (c) Quantitative RT-qPCR analysis of Tnfsf11 mRNA transcripts from the same samples used in the RNA-sequencing analysis for verification of Tnfsf11 suppression. sgRNA-3 cells compared to sgRNA-SV40 cells using Student’s t test. (d) Normalized expression values from the RNA-sequencing from UAMS-32 cells for the genes 1.5 Mb upstream (right side of plot) and downstream (left side of plot) from Tnfsf11. Relative location of Tnfsf11 is indicated with the labeled arrow. Adjusted p-value displayed for genes with p < 0.05. (e) Quantitative RT-qPCR analysis of Dnajc15 mRNA transcripts from UAMS-32 cells (sgRNA-3 versus sgRNA-SV40) and kidney from line 157 (n = 6) and their WT controls (n = 6). Both sexes were included. Comparisons were made using Student’s t test.
