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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 09, 2024 |
| Title |
CRISPRi with barcoded expression reporters dissects regulatory networks in human cells |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy and potential of CiBER-seq for dissecting genetic networks in mammalian cells with superior time resolution.
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| Overall design |
Sequencing of expressed barcode reporters from SFFV control promoters and synthetic NF-κB reporters, comparing the effects of CRISPRi induction (between matched SFFV promoters) and TNF-α treatment (between NF-κB and SFFV control)
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| Contributor(s) |
Kim J, Muller RY, Bondra ER, Ingolia NT |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 GM135233 |
High-precision pooled screening for quantitative molecular phenotypes |
UNIVERSITY OF CALIFORNIA BERKELEY |
Nicholas T Ingolia |
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| Submission date |
Aug 30, 2024 |
| Last update date |
Sep 09, 2024 |
| Contact name |
Nicholas T Ingolia |
| E-mail(s) |
ingolia@berkeley.edu, nick@ingolia.org
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| Phone |
510 664 7071
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| Organization name |
University of California, Berkeley
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| Department |
Molecular and Cell Biology
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| Lab |
Ingolia
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| Street address |
1 Barker Hall # 3202
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platforms (1) |
| GPL34281 |
Illumina NovaSeq X (Homo sapiens) |
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| Samples (30)
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| GSM8490353 |
mCherry, pre, rep1 |
| GSM8490354 |
iRFP, post, rep2 |
| GSM8490355 |
iRFP, pre, rep2 |
| GSM8490356 |
mCherry, post, rep2 |
| GSM8490357 |
mCherry, pre, rep2 |
| GSM8490358 |
iRFP, post, rep3 |
| GSM8490359 |
iRFP, pre, rep3 |
| GSM8490360 |
mCherry, post, rep3 |
| GSM8490361 |
mCherry, pre, rep3 |
| GSM8490362 |
iRFP, untreated, rep1 |
| GSM8490363 |
mCherry, unreated, rep1 |
| GSM8490364 |
iRFP, 2 hr TNFα, rep1 |
| GSM8490365 |
mCherry, 2 hr TNFα, rep1 |
| GSM8490366 |
iRFP, 5 hr TNFα, rep1 |
| GSM8490367 |
mCherry, 5 hr TNFα, rep1 |
| GSM8490368 |
iRFP, untreated, rep2 |
| GSM8490369 |
mCherry, untreated, rep2 |
| GSM8490370 |
iRFP, 2 hr TNFα, rep2 |
| GSM8490371 |
mCherry, 2 hr TNFα, rep2 |
| GSM8490372 |
iRFP, 5 hr TNFα, rep2 |
| GSM8490373 |
mCherry, 5 hr TNFα, rep2 |
| GSM8490374 |
iRFP, untreated, rep3 |
| GSM8490375 |
mCherry, untreated, rep3 |
| GSM8490376 |
iRFP, 2 hr TNFα, rep3 |
| GSM8490377 |
mCherry, 2 hr TNFα, rep3 |
| GSM8490378 |
iRFP, 5 hr TNFα, rep3 |
| GSM8490379 |
mCherry, 5 hr TNFα, rep3 |
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| Relations |
| BioProject |
PRJNA1154595 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE276055_NIJK03_iRFP_bccounts_final.txt.gz |
91.1 Mb |
(ftp)(http) |
TXT |
| GSE276055_NIJK03_mCherry_bccounts_final.txt.gz |
91.0 Mb |
(ftp)(http) |
TXT |
| GSE276055_NIJK04_iRFP_bccounts_final.txt.gz |
81.0 Mb |
(ftp)(http) |
TXT |
| GSE276055_NIJK04_iRFP_bccounts_reseq.txt.gz |
17.4 Mb |
(ftp)(http) |
TXT |
| GSE276055_NIJK04_mCherry_bccounts_final.txt.gz |
76.9 Mb |
(ftp)(http) |
TXT |
| GSE276055_NIJK04_mCherry_bccounts_reseq_final.txt.gz |
22.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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