A Pooled CRISPR Screen Protocol for Comparative Pathway Analysis Across Multiple Stressors
- 1University of Ottawa
Abstract
High content CRISPR screens have become a leading method for the identification of new cellular pathways, proteins functions, and drug targets. However, as the complexity of these screens increase, so does the possibility of artifacts. Here, we describe a refined CRISPR screening protocol that can compare acute cellular responses across multiple cellular treatments and timepoints, while reducing false positives by up to 40% compared to conventional screening approaches. This is achieved through the use of a fluorescence-based reporter line responsive to multiple stressors, as well as a fixation step to preserve acute response differences and avoid sorting-related false positives. We recently performed a version of this workflow to compare kinases regulating different types of selective autophagy, including starvation-induced autophagy, ERphagy, and pexophagy. Beyond autophagy research, this screen approach can be implemented for any fluorescence-based screen where preservation of acute cellular responses is important. Here we present a complete workflow of this optimized screening protocol using our selective autophagy screen as a specific example throughout. Implementation of these strategies should allow researchers to increase the number of stressors tested within a single screen without sacrificing cell health, reliability, or statistical power.
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BibTeX
@misc{losier2026pooled,
title = {{A Pooled CRISPR Screen Protocol for Comparative Pathway Analysis Across Multiple Stressors}},
author = {T. Losier and K. King and J. Taylor and M. Rousseaux and R. Russell},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.07.749902},
url = {https://www.biorxiv.org/content/10.64898/2026.09.07.749902v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - A Pooled CRISPR Screen Protocol for Comparative Pathway Analysis Across Multiple Stressors
AU - T. Losier
AU - K. King
AU - J. Taylor
AU - M. Rousseaux
AU - R. Russell
PY - 2026
DA - 2026/09/07
PB - bioRxiv
DO - 10.64898/2026.09.07.749902
UR - https://www.biorxiv.org/content/10.64898/2026.09.07.749902v1
AB - High content CRISPR screens have become a leading method for the identification of new cellular pathways, proteins functions, and drug targets. However, as the complexity of these screens increase, so does the possibility of artifacts. Here, we describe a refined CRISPR screening protocol that can compare acute cellular responses across multiple cellular treatments and timepoints, while reducing false positives by up to 40% compared to conventional screening approaches. This is achieved through the use of a fluorescence-based reporter line responsive to multiple stressors, as well as a fixation step to preserve acute response differences and avoid sorting-related false positives. We recently performed a version of this workflow to compare kinases regulating different types of selective autophagy, including starvation-induced autophagy, ERphagy, and pexophagy. Beyond autophagy research, this screen approach can be implemented for any fluorescence-based screen where preservation of acute cellular responses is important. Here we present a complete workflow of this optimized screening protocol using our selective autophagy screen as a specific example throughout. Implementation of these strategies should allow researchers to increase the number of stressors tested within a single screen without sacrificing cell health, reliability, or statistical power.
ER -