A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens
-
1.
California Institute of Technology
Abstract
Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, 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. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.
Copyright and License
© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Acknowledgement
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.
Contributions
K.P., A.G. J.R. and R.M.V. were responsible for conceptual design of the project. K.P., A.G., T.E., and M.W. performed experiments, K.P. and A.G. performed data analysis, and K.P., A.G., and R.M.V wrote the manuscript. R.M.V. and J.S.W. provided funding. All authors read and approved the final manuscript.
Funding
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 2019 L/LT000858 (AG), the Heritage Medical Research Institute (RMV), the NIH's National Institute of General Medical Sciences (DP2GM137412) (RMV), the Sontag Foundation (RMV), the Burroughs Wellcome Pathogenesis Investigator Program (RMV), NIH F31 Ruth L. Kirchstein National Research Service Award NS115380 (JMR), Rosen Family fellowship (KRP), and Arie Jan Haagen-Smit Fellowship (KRP).
Data Availability
All materials necessary for dual-guide construction are available via Addgene, with accession numbers listed in the Materials and Methods. All programmed sgRNA, reporter constructs and cell lines are available from the corresponding author upon request. The sequencing datasets generated during the current study are available in the Caltech DATA repository and can be publicly accessed, 10.22002/3hvyj-yzq30.
Conflict of Interest
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. No potential conflicts of interests were declared by the other authors.
Data Availability
All materials necessary for dual-guide construction are available via Addgene, with accession numbers listed in the Materials and Methods. All programmed sgRNA, reporter constructs and cell lines are available from the corresponding author upon request. The sequencing datasets generated during the current study are available in the Caltech DATA repository and can be publicly accessed, 10.22002/3hvyj-yzq30.
Files
Additional details
- PMCID
- PMC10614335
- Howard Hughes Medical Institute
- 2RM1 HG009490-06
- National Institutes of Health
- 2019L/LT000858
- Human Frontier Science Program
- Heritage Medical Research Institute
- DP2GM137412
- National Institutes of Health
- Sontag Foundation
- Burroughs Wellcome Fund
- F31 NS115380
- National Institutes of Health
- Rosen Family fellowship
- Arie Jan Haagen-Smit Fellowship
- Caltech groups
- Tianqiao and Chrissy Chen Institute for Neuroscience, Heritage Medical Research Institute, Division of Biology and Biological Engineering