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

Alina Guna; Katharine R Page; Joseph M Replogle; Theodore K Esantsi; Maxine L Wang; Jonathan S Weissman; Rebecca M Voorhees

doi:10.1186/s12864-023-09754-y

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

BMC Genomics. 2023 Oct 30;24(1):651. doi: 10.1186/s12864-023-09754-y.

Authors

Alina Guna^#^{1

2}, Katharine R Page^#¹, Joseph M Replogle^{2

3

4}, Theodore K Esantsi^{2

4}, Maxine L Wang^{1

2}, Jonathan S Weissman^{2

4

5

6}, Rebecca M Voorhees^{7

8}

Affiliations

¹ Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Ave, Pasadena, CA, 91125, USA.
² Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
³ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, 94158, USA.
⁴ Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
⁵ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
⁶ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA.
⁷ Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Ave, Pasadena, CA, 91125, USA. voorhees@caltech.edu.
⁸ Howard Hughes Medical Institute Freeman Hrabowski Scholar, California Institute of Technology, Pasadena, CA, 91125, USA. voorhees@caltech.edu.

^# Contributed equally.

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.

Keywords: CRISPR interference; ER membrane protein complex; Epistasis; Genetic modifier; Genome-wide screen; Tail-anchored proteins.

MeSH terms

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats*
Gene Library
Genome
RNA, Guide, CRISPR-Cas Systems*

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

MeSH terms

Substances

Grants and funding