Rapid Multiplexed Flow Cytometric Validation of CRISPRi sgRNAs in Tissue Culture

John S Chorba; Vivian Q Xia; Geoffrey A Smith; Arun Padmanabhan

doi:10.21769/BioProtoc.4591

Rapid Multiplexed Flow Cytometric Validation of CRISPRi sgRNAs in Tissue Culture

Bio Protoc. 2023 Jan 20;13(2):e4591. doi: 10.21769/BioProtoc.4591.

Authors

John S Chorba^{1

2

3}, Vivian Q Xia^{1

2

3}, Geoffrey A Smith^{1

2

3}, Arun Padmanabhan^{1

2

3}

Affiliations

¹ Division of Cardiology, Zuckerberg San Francisco General Hospital, San Francisco CA, USA.
² Department of Medicine, University of California San Francisco, San Francisco CA, USA.
³ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco CA, USA.

Abstract

Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems. Graphical abstract.

Keywords: CRISPRi; Flow cytometry; Forward genetics; Hit validation.

Abstract

Grants and funding