Valentina Diehl, Martin Wegner, Paolo Grumati, Koraljka Husnjak, Simone Schaubeck, Andrea Gubas, Varun Jayeshkumar Shah, Ibrahim H. Polat, Felix Langschied, Cristian Prieto García, Konstantin Müller, Alkmini Kalousi, Ingo Ebersberger, Christian Hubertus Brandts, Ivan Đikić, Manuel Kaulich
- 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.
MetadatenAuthor: | Valentina DiehlGND, Martin WegnerORCiD, Paolo GrumatiORCiDGND, Koraljka HusnjakORCiD, Simone Schaubeck, Andrea GubasORCiD, Varun Jayeshkumar Shah, Ibrahim H. Polat, Felix Langschied, Cristian Prieto GarcíaORCiDGND, Konstantin Müller, Alkmini KalousiORCiD, Ingo EbersbergerORCiDGND, Christian Hubertus BrandtsORCiDGND, Ivan ĐikićORCiDGND, Manuel KaulichORCiD |
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URN: | urn:nbn:de:hebis:30:3-737722 |
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DOI: | https://doi.org/10.1093/nar/gkab309 |
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ISSN: | 1362-4962 |
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Parent Title (English): | Nucleic acids research |
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Publisher: | Oxford Univ. Press |
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Place of publication: | Oxford |
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Document Type: | Article |
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Language: | English |
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Date of Publication (online): | 2021/05/06 |
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Date of first Publication: | 2021/05/06 |
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Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
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Release Date: | 2023/05/09 |
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Volume: | 49 |
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Issue: | 10 |
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Page Number: | 21 |
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First Page: | 5684 |
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Last Page: | 5704 |
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Note: | 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] |
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Note: | 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). |
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Institutes: | Biowissenschaften |
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| Medizin |
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| Angeschlossene und kooperierende Institutionen / Senckenbergische Naturforschende Gesellschaft |
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| Fachübergreifende Einrichtungen / Biodiversität und Klima Forschungszentrum (BiK-F) |
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| Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) |
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Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
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| 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
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Sammlungen: | Universitätspublikationen |
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Licence (German): | Creative Commons - CC BY-NC - Namensnennung - Nicht kommerziell 4.0 International |
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