Fig. 4: CRISPR-TRAPseq-mediated QKI deletion in maturing astrocytes alters target transcripts.

A Cartoon strategy of QKI knockdown in cortical astrocytes. Brain icon is used under Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/). B Transduced CFP-positive cells (detected by GFP antibody) from GFAP:CFP QKI gRNA-injected mice (Cas9−(top) and Cas9+(bottom)) reveal loss of QKI-6 immunoreactivity in Cas9+ animals, but not in Cas9− animals or in non-transduced adjacent cells. Scale bar is 20 µm. C Quantification of QKI pixels in CFP+ cells across genotypes reveals median 79% reduction in Cas9+ cells. Total pixel number per cell was normalized to WT littermates’ values. Median value within genotypes is represented as large black circle. P value represents results of a linear mixed model to account for random effects of individual animals. N = 270 WT, and 280 Cas9+ cells from 3 WT animals and 8 Cas9 animals. Source data are provided as a Source Data file. D Volcano plot showing mRNA enriched by TRAP from GFAP-Cre gRNA transduced P21 astrocytes is highly enriched in previously defined markers of astrocytes (red) and depleted in markers on neurons (green)⁵⁶, confirming method. P-values are generated from likelihood ratio tests (LRT) in this contrast using edgeR. E TRAP analysis identifies transcripts with significantly altered ribosomal association in vivo following QKI mutation (blue, increased in WT, yellow increased in QKI KO. P values are generated from likelihood ratio tests (LRT) in this contrast using edgeR. F Individual genes with altered ribosomal association following QKI deletion. Data are represented by boxplots (center = median, minima and maxima represented by whisker ends, and box bounds representing the 25th and 75th percentiles of the data). The p value in each plot represent two-sided Student’s t test. G Heatmap of all TRAP and Input samples showing transcripts with altered ribosomal association. H Forest plot illustrates overlap between QKI responsive transcripts, CLIP genes, and locally translated genes as defined⁵. The odds ratio represents one-sided Fisher’s exact test. The whiskers represent 95% confidence interval for the odds ratio, and the number of genes in each group are indicated in parentheses above the bars. I Cumulative distribution function (CDF) and boxplot (J) of CLIP targets (purple) expressed in astrocytes show a decrease in QKI CLIP target transcript levels after QKI deletion (two-sided Student’s t test, p value = 2.9e−12). Data are represented by boxplots (center = median, minima and maxima represented by whisker ends, and box bounds representing the 25th and 75th percentiles of the data).