Combinatorial Genetic Screening in Cancer

Abstract

Chromatin-modifying enzymes impact cell state and function. They are frequently mutated in disease, including many cancers, and present promising opportunities for therapeutic intervention. These enzymes are often found in multi-subunit complexes; over 500 chromatin-associated proteins form over 160 chromatin-modifying complexes. These complexes possess diverse enzymatic activities and binding domains and display various allosteric regulatory mechanisms. Faithful tools to elucidate relationships within and between these complexes in mammalian cells have been lacking. We developed a CRISPR-Cas9 based platform for combinatorial screening of genetic relationships, and the genetic backgrounds and cellular contexts in which they exist. The combinatorial platform termed “Big Papi” uses two orthologous Cas9 enzymes to promote independent Cas9 loading and targeting. The technology was validated in six cancer cell lines with two independent libraries targeting well-known synthetic lethal gene pairs. Furthermore, we demonstrate that our system is useful for bimodal applications, such as simultaneous CRISPR-activation and CRISPR cutting. Next, the Big Papi system was extended and applied to screen over 300,000 combinatorial interactions of chromatin regulators in leukemia. This identified core as well as cell type-specific synthetic lethal combinations. Overall, our findings uncover critical chromatin regulator interactions and inform synthetic lethal dependencies that can be targeted in cancer.