TY  - JOUR
A1  - Diehl, Valentina
A1  - Wegner, Martin
A1  - Grumati, Paolo
A1  - Husnjak, Koraljka
A1  - Schaubeck, Simone
A1  - Gubas, Andrea
A1  - Shah, Varun Jayeshkumar
A1  - Polat, Ibrahim H.
A1  - Langschied, Felix
A1  - Prieto García, Cristian
A1  - Müller, Konstantin
A1  - Kalousi, Alkmini
A1  - Ebersberger, Ingo
A1  - Brandts, Christian Hubertus
A1  - Đikić, Ivan
A1  - Kaulich, Manuel
T1  - Minimized combinatorial CRISPR screens identify genetic interactions in autophagy
T2  - Nucleic acids research
N2  - Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/73772
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-737722
SN  - 1362-4962
N1  - Funding:
Hessisches Ministerium für Wissenschaft und Kunst (HMWK) [LOEWE-CGT IIIL5-518/17.004, LOEWE-FCI IIIL5-519/03/03.001]; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [CEF-MC-EXC115/2, ECCPS-EXC147/2, CPI-EXC 2026, 259130777 – SFB 1177]. Funding for open access charge: DFG [SFB 1177 - 259130777]
N1  - Sequencing data are provided as raw read count tables as Supplementary Tables S2-S5. RNAseq data are accessible from GEO (GSE166851). Plasmids encoding extended- and combinatorial autophagy libraries can be requested through the Goethe University

Depository (https://innovectis.de/en/technologies/goethe-depository/, please select ‘Minimized combinatorial CRISPR screens identify genetic interactions in autophagy’) or directly contact the Corresponding Author. Custom scripts are available from GitHub (https://github.com/ManuelKaulich/3Cs-multiplexing).
VL  - 49
IS  - 10
SP  - 5684
EP  - 5704
PB  - Oxford Univ. Press
CY  - Oxford
ER  -