Combinatorial CRISPR screening reveals functional buffering in autophagy

Abstract

Functional genomics studies in model organisms and human cell lines provided important insights into gene functions and their context-dependent role in genetic circuits. However, our functional understanding of many of these genes and how they combinatorically regulate key biological processes, remains limited. To enable the SpCas9-dependent mapping of gene-gene interactions in human cells, we established 3Cs multiplexing for the generation of combinatorial gRNA libraries in a distribution-unbiased manner and demonstrate its robust performance. The optimal number for combinatorial hit calling was 16 gRNA pairs and the skew of a library’s distribution was identified as a critical parameter dictating experimental scale and data quality. Our approach enabled us to investigate 247,032 gRNA-pairs targeting 12,736 gene-interactions in human autophagy. We identified novel genes essential for autophagy and provide experimental evidence that gene-associated categories of phenotypic strengths exist in autophagy. Furthermore, circuits of autophagy gene interactions reveal redundant nodes driven by paralog genes. Our combinatorial 3Cs approach is broadly suitable to investigate unexpected gene-interaction phenotypes in unperturbed and diseased cell contexts.

Functional Genomics Approaches to Elucidate Vulnerabilities of Intrinsic and Acquired Chemotherapy Resistance

“…More often, several co-occurring gain-of-function and/or loss-of-function mechanisms synergistically contribute to a patient’s chemoresistance profile. In this context, technologies for the robust performance of genetic multiplexing approaches for the simultaneous perturbation of multiple loci are needed [ 73 , 216 ]. While these combinatorial approaches gain momentum in the identification of synthetic gene interactions, their general application and analysis are far from standardized.…”