Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

  • Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

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Author:Martin Wegner, Valentina Diehl, Verena Bittl, Rahel de Bruyn, Svenja Wiechmann, Yves Matthess, Marie Hebel, Michael G. B. Hayes, Simone Schaubeck, Christopher Benner, Sven Heinz, Anja Bremm, Ivan ĐikićORCiDGND, Andreas Ernst, Manuel KaulichORCiD
Pubmed Id:
Parent Title (English):eLife
Publisher:eLife Sciences Publications
Place of publication:Cambridge
Contributor(s):Jonathan Weissman
Document Type:Article
Year of Completion:2019
Year of first Publication:2019
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/04/02
Tag:3Cs technology; Crispr/Cas; DUBs; Doxorubicin; Tools and ressources; cell biology; gRNA library; genetics and genomics; genome-wide; human
Page Number:31
First Page:1
Last Page:31
Copyright Wegner et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Institutes:Biowissenschaften / Biowissenschaften
Medizin / Medizin
Exzellenzcluster / Exzellenzcluster Makromolekulare Komplexe
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung 4.0