Abstract Pairwise perturbation of gene function using the CRISPR/Cas9 system has huge potential in screening for genetic interactions and synthetic lethal gene pairs to identify novel combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs, establishing our method for performing efficient large scale genetic interaction screens. This system is versatile and can be used with most guide RNA vector systems, and for other uses of paired guide delivery such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.

Genetic interaction library screening with a next-generation dual guide CRISPR system