CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes
Ryan Muller1, Zuriah A Meacham1, Lucas Ferguson1
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Science (New York, N.Y.)
|December 11, 2020
Summary
We developed CiBER-seq to measure gene function across genome-wide libraries. This CRISPR interference (CRISPRi) method connects genetic perturbations to specific molecular outcomes, advancing genetic network dissection.
Area of Science:
- Molecular Biology
- Genetics
- Systems Biology
Background:
- CRISPR-Cas9 technology enables genome-wide genetic screens, but quantifying molecular phenotypes remains a challenge.
- Existing methods struggle to link specific genetic perturbations to their precise molecular consequences across large libraries.
- Understanding complex biological pathways requires tools that can profile multiple molecular levels simultaneously.
Purpose of the Study:
- To develop and validate a novel method, CiBER-seq, for high-throughput, genome-wide profiling of molecular phenotypes.
- To connect CRISPR interference (CRISPRi) genetic perturbations to transcriptional, translational, and posttranslational reporter outputs.
- To apply CiBER-seq to dissect the integrated stress response (ISR) pathway in yeast.
Main Methods:
- CRISPR interference (CRISPRi) coupled with barcoded expression reporter sequencing (CiBER-seq) was employed.
- A pooled library approach allowed for simultaneous screening of numerous genetic perturbations.
- Reporter gene expression at transcriptional, translational, and posttranslational levels was quantified via barcoded sequencing.
Main Results:
- CiBER-seq successfully recapitulated the known integrated stress response (ISR) pathway in yeast.
- Genetic perturbations leading to uncharged transfer RNA (tRNA) accumulation were shown to activate ISR reporter transcription.
- The study identified novel triggers for ISR activation, including tRNA insufficiency independent of the canonical sensor.
Conclusions:
- CiBER-seq is a powerful and broadly applicable tool for dissecting complex genetic networks.
- The method enables precise, comprehensive profiling of molecular phenotypes from genome-wide genetic perturbations.
- This approach advances our understanding of gene function and pathway regulation, exemplified by novel insights into the ISR pathway.
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