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| Item Type: | Dataset |
|---|---|
| Title: | Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [bulkCRISPR-screen] |
| Creators Name: | Vučićević, D., Hsu, C.W., Lopez Zepeda, L.S., Burkert, M., Hirsekorn, A., Bilić, I., Kastelić, N., Landthaler, M., Lacadie, S.A. and Ohler, U. |
| Abstract: | ORGANISMS: Homo sapiens; synthetic construct. 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: CRISPR activation or CRISPR inhibition screen in the 2 MB genomic space around PHOX2B was performed in SHSY-5Y cell line. Cells were collected and various time points. Oligo pools containing 46,722 oligos (Table S3) ordered from Twist Bioscience were cloned into the CROPseq-Guide-Puro plasmid (Addgene #86708) digested with Esp3I (Thermo Fisher Scientific #FD0454) according to (Joung et al., 2017b). The library was amplified using primers and according to the protocol from (Datlinger et al., 2017) and sequenced on a NextSeq 500/550 machine according to the Illumina user manual. Lentiviral production was done as described in (Datlinger et al., 2017). The lentiviral prep was concentrated using Lenti-X Concentrator (Takara Bio #631232) and the viral titer was determined using a Crystal violet viability assay (Vučićević et al., 2016). The screen was performed as described in (Datlinger et al., 2017). For each gRNA at least 1000 cells per gRNA were seeded and they were transduced at a low multiplicity of infection (<0.3 MOI) in either SH-SY5Y-dCas9VPR (CRISPRa) and SH-SY5Y-dCas9KRAB (CRISPRi) cell line. The cells were selected 48h post-transduction for the ones that received the gRNA using 2ug/ml of puromycin (Sigma Aldrich #P9620) for three days. They were kept under constant antibiotic selection with 1ug/ml of puromycin and 100ug/ml of hygromycin and collected at several time points (day 5, day 20, day 29 and day 33). DNA was extracted using the Quick-DNA Midiprep Plus kit (Zymo Research #D4075). The library preparation was done as described in (Datlinger et al., 2017) and sequenced on a NextSeq 500/550 according to the Illumina protocol. |
| Source: | Gene Epression Omnibus (GEO) |
| Publisher: | NCBI |
| Date: | 11 September 2025 |
| Official Publication: | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274255 |
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