Fig. 4.

CRISPRi of antisense transcription initiation at neural crest genes. (A) UCSC Genome Browser snapshot of the sox9a and foxd3 loci with ATAC-seq (black track) and strand-specific RNA-seq (red track, sense; blue track, antisense) from sox10+ cells (ATAC-seq) or nuclei (RNA-seq). Selected guide RNAs target the initiation of antisense transcription (star) of sox9a-AS or foxd3-AS. (A′) Hybridisation Chain Reaction detection of coding/sense (sox9a, foxd3) and antisense (sox9a-AS, foxd3-AS) transcripts in 24 hpf embryos. Antisense transcripts/puncta (cyan) were expressed in a ubiquitous, basal-like fashion. A similar basal-like expression of sense transcripts/puncta (red channel) was observed, along with spatially restricted regions of higher expression (white arrows). Single plane confocal images of the otic vesicle region are shown. Scale bars: 50 µm. (B) Sox10:CRISPRi workflow to investigate effect of sense/antisense transcription in microinjected zebrafish embryos. Ac/Ds U6 cloned guides were pooled according to experimental design (10 guides in total, five per locus) and transformed into bacteria to obtain a single prep per pool of high quality. Guide pools (foxd3+sox9a; or scrambled) were microinjected into one-cell-stage ox117 embryos and allowed to develop for 24 h. Embryos were dissociated and FAC-sorted to collect sox10:Citrine+ cells expressing dCas9-SID4x. RNA was extracted and RNAseq libraries were prepared using rRNA-depletion followed by strand-specific dUTP method with two replicates per condition. Transcript quantification and differential expression were performed using the kallisto/sleuth statistical pipeline. (C) Quantification of transcripts (transcripts per million, TPM) following sox10:CRISPRi. Bootstrapped estimates of sense and antisense sox9a/foxd3 transcripts in each biological replicate. Down/upregulation could be detected for sense and antisense foxd3/sox9a transcripts in experimental versus scrambled condition although not statistically significant, with the exception of foxd3 (FC=1.261, qval=0.007). (C′) PANTHER Gene Ontology (GO) overrepresentation statistical analysis of differentially expressed genes (qval <0.05). Top 10 GO terms (Fisher's exact test, with false discovery rate <0.01) shown.