Journal Article

DKFZ-2020-01622

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.

Becker, M. ; Noll-Puchta, H. ; Amend, D. ; Nolte, F. ; Fuchs, C. ; Jeremias, I.DKFZ* ; Braun, C. J. (Last author)DKFZ*

2020
Oxford Univ. Press Oxford

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Please use a persistent id in citations: doi:10.1093/nar/gkaa459

Abstract: 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.

Note: #LA:B062#

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Contributing Institute(s):

1. DKTK MU LMU zentral (MU01)
2. B062 Pädiatrische Neuroonkologie (B062)

Research Program(s):

1. 312 - Functional and structural genomics (POF3-312) (POF3-312)

Appears in the scientific report 2020

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