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| Item Type: | Dataset |
|---|---|
| Title: | Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [TESLA-seq] |
| Creators Name: | Vučićević, Dubravka, Hsu, C.W., Lopez Zepeda, L.S., Burkert, M., Hirsekorn, A., Bilić, I., Kastelić, N., Landthaler, M., Lacadie, S.A. and Ohler, U. |
| Abstract: | ORGANISM: Homo sapiens. EXPERIMENT TYPE: Other. SUMMARY: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system. OVERALL DESIGN: TESLA-seq was performed as described in details in the manuscript. We selected 222 top-scoring significant CaREs from the bulk phenotypic screen. For each of them, we selected gRNAs with the highest fold change. These gRNAs, together with 52 control gRNAs (total of 1098 in Table S5), were ordered as oligos from Twist Bioscience, cloned, and sequenced as described for the phenotypic screen. The lentiviral production was done as in the viability screen. SHSY-5Y-VPR line was infected at <0.3 MOI and the selection with 2ug/ul of puromycin started 24h post transduction. As soon as the antibiotic control non-infected cells were dead,on day 4 after transduction, the cells were collected. The viability of the cells was > 85% in both experiments as determined by the BD Rhapsody scanner after staining with Calcein AM (Thermo Fisher Scientific #C1430) and Draq7 (Thermo Fisher Scientific #564904) according to the manufacturer's protocol. Single-cell capture and cDNA synthesis were performed using the BD RhapsodyTM Single-Cell Analysis System according to the manufacturer's instructions. Capture probes for 146 transcripts corresponding to 78 genes in the 6MB genomic space (+/- 3MB from the PHOX2B TSS) on chr4 were designed by BD. The targets were enriched and the library was prepared according to the BD mRNA Targeted Library Preparation protocol. Paired-end sequencing (2x75nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles with a 20% PhiX spike-in. Two replicates of this experiment are submited: TESLA-seq replicate_1 and TESLA-seq_replicate_2. |
| Source: | Gene Epression Omnibus (GEO) |
| Publisher: | NCBI |
| Date: | 11 September 2025 |
| Official Publication: | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274256 |
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