Gene

Biology

Cas9

Directionality

Gene knockout

CRISPR

Gene regulatory network

Computational biology

Genetics

Genetic network

Gene expression

Phenotype

Nature Biotechnology

Combining gene activation and knockout of different genes in the same cell using two different Cas9 enzymes enables the reconstruction of directional dependency. Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.

Dual gene activation and knockout screen reveals directional dependencies in genetic networks