Home > Publications database > CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries. > print

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024 7 _ |a 10.1093/nar/gkaa459
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041 _ _ |a eng
082 _ _ |a 570
100 1 _ |a Becker, Martin
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245 _ _ |a CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.
260 _ _ |a Oxford
|c 2020
|b Oxford Univ. Press
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520 _ _ |a 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.
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700 1 _ |a Noll-Puchta, Heidi
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700 1 _ |a Amend, Diana
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700 1 _ |a Nolte, Florian
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700 1 _ |a Fuchs, Christiane
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700 1 _ |a Jeremias, Irmela
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700 1 _ |a Braun, Christian Jörg
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773 _ _ |a 10.1093/nar/gkaa459
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