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Genome-scale CRISPR screening in a single mouse liver

View ORCID ProfileHeather R. Keys, View ORCID ProfileKristin A. Knouse
doi: https://doi.org/10.1101/2021.01.30.428976
Heather R. Keys
1Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA
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Kristin A. Knouse
2Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
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  • For correspondence: knouse@mit.edu
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SUMMARY

A complete understanding of the genetic determinants underlying mammalian physiology and disease is limited by the availability of tools for high-throughput genetic dissection in the living organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for uncovering the genetic regulation of cellular processes, but the need to stably deliver single guide RNAs to millions of cells has largely restricted its implementation to ex vivo systems. There thus remains a pressing need for accessible high-throughput functional genomics in a living organism. Here, we establish genome-wide enrichment and depletion screening in the liver of a single mouse and use this approach to uncover the complete regulation of cellular fitness in a living organism. We uncover regulation of cell fitness not previously identified in cell culture screens, underscoring the power of bringing high-throughput functional genomics into the organism. The approach we developed is accessible, robust, scalable, and adaptable to diverse phenotypes and CRISPR applications. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into physiology and disease.

Competing Interest Statement

H.R.K. and K.A.K. are co-inventors on a patent filed by the Whitehead Institute related to work in this manuscript.

Footnotes

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  • This version of the manuscript has been revised to include further validation that the screening approach does not lead to hepatocyte damage or liver inflammation (Supplemental Figure 2E) and additional analyses demonstrating that positive and negative regulators of a phenotype can be uncovered in single mice (Supplemental Figure 3G). Additionally, we have replaced CRISPR-based validation of hits with orthogonal validation approaches and further explore and confirm the requirement for heparan sulfate biosynthesis in hepatocytes (Figure 5E, 5F, Supplemental Figure 5D, 5E).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted August 28, 2022.
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Genome-scale CRISPR screening in a single mouse liver
Heather R. Keys, Kristin A. Knouse
bioRxiv 2021.01.30.428976; doi: https://doi.org/10.1101/2021.01.30.428976
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Genome-scale CRISPR screening in a single mouse liver
Heather R. Keys, Kristin A. Knouse
bioRxiv 2021.01.30.428976; doi: https://doi.org/10.1101/2021.01.30.428976

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