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Elife. 2019 Mar 6;8. pii: e42549. doi: 10.7554/eLife.42549.

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

Author information

1
Institute of Biochemistry II, Goethe University Frankfurt - Medical Faculty, University Hospital, Frankfurt, Germany.
2
Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany.
3
Project Group Translational Medicine & Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt, Germany.
4
Department of Medicine, University of California, San Diego, San Diego, United States.
5
Frankfurt Cancer Institute, Frankfurt am Main, Germany.
6
Cardio-Pulmonary Institute, Frankfurt am Main, Germany.
#
Contributed equally

Abstract

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.

KEYWORDS:

3Cs technology; CRISPR/Cas; DUBs; Doxorubicin; cell biology; gRNA library; genetics; genome-wide; genomics; human

Conflict of interest statement

MW The Goethe University Frankfurt has filed a patent related to this work on which MW is an inventor (WO2017EP84625), VD The Goethe University Frankfurt has filed a patent related to this work on which VD is an inventor (WO2017EP84625), VB, YM, MH, MH, SS, CB, SH, AB No competing interests declared, Rd The Goethe University Frankfurt has filed a patent related to this work on which RDB is an inventor (WO2017EP84625), SW The Goethe University Frankfurt has filed a patent related to this work on which SW is an inventor (WO2017EP84625), ID is co-founder, shareholder and CEO of Vivlion GmbH in Gründung. Also a senior editor of eLife, AE The Goethe University Frankfurt has filed a patent related to this work on which AE is an inventor (WO2017EP84625), MK The Goethe University Frankfurt has filed a patent related to this work on which MK is an inventor (WO2017EP84625). Also co-founder, shareholder and CSO of Vivlion GmbH in Gründung

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