%0 Journal Article
%A Becker, Martin
%A Noll-Puchta, Heidi
%A Amend, Diana
%A Nolte, Florian
%A Fuchs, Christiane
%A Jeremias, Irmela
%A Braun, Christian Jörg
%T CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.
%J Nucleic acids research
%V 48
%N 13
%@ 0301-5610
%C Oxford
%I Oxford Univ. Press
%M DKFZ-2020-01622
%P e78
%D 2020
%Z #LA:B062#
%X 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.
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:32479629
%2 pmc:PMC7367185
%R 10.1093/nar/gkaa459
%U https://inrepo02.dkfz.de/record/157427