TY  - JOUR
A1  - Wegner, Martin
A1  - Diehl, Valentina
A1  - Bittl, Verena
A1  - Bruyn, Rahel de
A1  - Wiechmann, Svenja
A1  - Matthess, Yves
A1  - Hebel, Marie
A1  - Hayes, Michael G. B.
A1  - Schaubeck, Simone
A1  - Benner, Christopher
A1  - Heinz, Sven
A1  - Bremm, Anja
A1  - Đikić, Ivan
A1  - Ernst, Andreas
A1  - Kaulich, Manuel
T1  - Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome
T2  - eLife
N2  - 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.
KW  - Tools and ressources
KW  - cell biology
KW  - genetics and genomics
KW  - Crispr/Cas
KW  - gRNA library
KW  - 3Cs technology
KW  - genome-wide
KW  - DUBs
KW  - Doxorubicin
KW  - human
Y1  - 2019
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/48808
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-488081
SN  - 2050-084X
N1  - 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.
VL  - 8
IS  - e42549
SP  - 1
EP  - 31
PB  - eLife Sciences Publications
CY  - Cambridge
ER  -