TY  - JOUR
AU  - Becker, Martin
AU  - Noll-Puchta, Heidi
AU  - Amend, Diana
AU  - Nolte, Florian
AU  - Fuchs, Christiane
AU  - Jeremias, Irmela
AU  - Braun, Christian Jörg
TI  - CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.
JO  - Nucleic acids research
VL  - 48
IS  - 13
SN  - 0301-5610
CY  - Oxford
PB  - Oxford Univ. Press
M1  - DKFZ-2020-01622
SP  - e78
PY  - 2020
N1  - #LA:B062#
AB  - The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.
LB  - PUB:(DE-HGF)16
C6  - pmid:32479629
C2  - pmc:PMC7367185
DO  - DOI:10.1093/nar/gkaa459
UR  - https://inrepo02.dkfz.de/record/157427
ER  -