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CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

Ryan Muller, Zuriah A. Meacham, Lucas Ferguson, View ORCID ProfileNicholas T. Ingolia
doi: https://doi.org/10.1101/2020.03.29.015057
Ryan Muller
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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Zuriah A. Meacham
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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Lucas Ferguson
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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Nicholas T. Ingolia
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA
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  • ORCID record for Nicholas T. Ingolia
  • For correspondence: ingolia@berkeley.edu
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Abstract

To realize the promise of CRISPR/Cas9-based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We address this challenge by profiling transcriptional, translational, and post-translational reporters using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach connects an entire library of guides to their individual phenotypic consequences using pooled sequencing. We show that CiBER-seq profiling fully recapitulates the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged tRNA accumulation activated ISR reporter transcription. Surprisingly, tRNA insufficiency also activated the reporter, independent of the Gcn2 kinase that senses uncharged tRNAs. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.

Footnotes

  • Added reference to contemporaneous study employing similar approach: B. D. Alford, G. Valiant, O. Brandman, Genome-wide, time-sensitive interrogation of the heat shock response under diverse stressors via ReporterSeq. bioRxiv, doi.org:10.1101/2020.03.29.014845 (2020).

  • https://ingolia-lab.github.io/CiBER_seq

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted March 31, 2020.
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CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes
Ryan Muller, Zuriah A. Meacham, Lucas Ferguson, Nicholas T. Ingolia
bioRxiv 2020.03.29.015057; doi: https://doi.org/10.1101/2020.03.29.015057
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CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes
Ryan Muller, Zuriah A. Meacham, Lucas Ferguson, Nicholas T. Ingolia
bioRxiv 2020.03.29.015057; doi: https://doi.org/10.1101/2020.03.29.015057

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