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A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Guna, Alina and Page, Katharine R. and Replogle, Joseph R. and Esantsi, Theodore K. and Wang, Maxine L. and Weissman, Jonathan S. and Voorhees, Rebecca M. (2023) A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens. . (Unpublished)

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The ability to map genetic interactions has been essential for determining gene function and defining biological pathways. Therefore, a system to readily perform genome-wide genetic modifier screens in human cells is a powerful platform for dissecting complex processes in mammalian cells, where redundancy and adaptation commonly mask the phenotype of a single genetic perturbation. Here, we report a CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, that can be used to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. As a proof of principle, we apply our approach to study the pathways that mediate tail-anchored (TA) protein insertion at the endoplasmic reticulum (ER). We show that this dual-guide library approach can be successfully coupled with FACS-based reporter screening, to identify genetic epistasis and thereby place TA biogenesis factors in their respective parallel pathways. We demonstrate that this dual-guide approach is both more sensitive and specific than traditional growth screening approaches, and is ideally suited for dissecting the complex interplay between factors in human cells.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Guna, Alina0000-0003-0757-1255
Replogle, Joseph R.0000-0003-1832-919X
Wang, Maxine L.0000-0002-5285-1857
Weissman, Jonathan S.0000-0003-2445-670X
Voorhees, Rebecca M.0000-0003-1640-2293
Additional Information: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-NC-ND 4.0 International license. We thank K. Hickey, R. Saunders, K. Popova and A. Inglis for helpful discussions. We thank the Whitehead Institute Flow Cytometry Core access to FACS machines and flow cytometers, and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech for sequencing of screening libraries. Research reported in this publication was supported by: Howard Hughes Medical Institute (JSW), Center for Genome Editing and Recording 2RM1 HG009490-06 (JSW), Human Frontier Science Program 2019L/LT000858 (AG), the Heritage Medical Research Institute (RMV), the NIH’s National Institute of General Medical Sciences (DP2GM137412) (RMV), NIH F31 Ruth L. Kirchstein National Research Service Award NS115380 (JMR), Rosen Family fellowship (KRP), and Arie Jan Haagen-Smit Fellowship (KRP). Competing Interest Statement. JMR consults for Maze Therapeutics and Waypoint Bio. JSW declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics, and Third Rock Ventures. RMV is a consultant and equity holder in Gate Bioscience. The Regents of the University of California with JSW as inventor have filed patent applications related to CRISPRi/a screening and Perturb-seq. JSW is an inventor on US Patent 11,254,933 related to CRISPRi/a screening.
Group:Millard and Muriel Jacobs Genetics and Genomics Laboratory, Heritage Medical Research Institute
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH2RM1 HG009490-06
Human Frontier Science Program2019L/LT000858
Heritage Medical Research InstituteUNSPECIFIED
NIH Postdoctoral FellowshipF31 NS115380
Caltech Division of Biology and Biological EngineeringUNSPECIFIED
Record Number:CaltechAUTHORS:20230316-182439000.33
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:120148
Deposited By: George Porter
Deposited On:20 Mar 2023 20:21
Last Modified:20 Mar 2023 20:21

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