Methods in molecular biology (Clifton, N.J.)Sep 30, 2021

Finding Gene Pairs That Cause Cell Death Using Large-Scale CRISPR Testing

Updated Apr 2, 2026

Abstract

A high-throughput method can identify synthetic lethal interactions (SLIs) using CRISPR, potentially covering hundreds of gene pairs.

Synthetic lethality explores genetic relationships between gene pairs to selectively target cancer cells.
Pooled screening formats used for identifying SLIs require extensive validation to reduce false positives.
The described method integrates plasmid DNA preparation, lentiviral production, and cellular transduction with single guide RNAs.
Image-based quantification enables high-resolution comparison of SLIs on an individual basis.
This scalable CRISPR screening approach can be applied to various cancer cell types and genes of interest.

Simplified

Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.

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