Combinatorial CRISPR screening reveals functional buffering in autophagy

  • 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.

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Author:Valentina DiehlGND, Martin WegnerORCiD, Paolo GrumatiORCiDGND, Koraljka HusnjakORCiD, Simone Schaubeck, Andrea GubasORCiD, Varun Jayeshkumar Shah, Felix Langschied, Alkmini KalousiORCiD, Ingo EbersbergerORCiDGND, Ivan ĐikićORCiDGND, Manuel KaulichORCiD
Parent Title (English):bioRxiv
Document Type:Preprint
Date of Publication (online):2020/07/26
Date of first Publication:2020/07/26
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/07/18
Page Number:45
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International