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CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries

View ORCID ProfileMartin Becker, Heidi Noll-Puchta, Diana Amend, Florian Nolte, Christiane Fuchs, Irmela Jeremias, Christian J Braun
doi: https://doi.org/10.1101/2020.04.02.021279
Martin Becker
1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Center for Environmental Health (HMGU), 81377 Munich, Germany
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  • ORCID record for Martin Becker
Heidi Noll-Puchta
2Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University of Munich (LMU), 81377 Munich, Germany
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Diana Amend
1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Center for Environmental Health (HMGU), 81377 Munich, Germany
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Florian Nolte
3Faculty of Business Administration and Economics, Bielefeld University, Bielefeld, Germany
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Christiane Fuchs
3Faculty of Business Administration and Economics, Bielefeld University, Bielefeld, Germany
4Department of Mathematics, Technische Universität München, Munich, Germany
5Helmholtz Zentrum München - German Research Center for Environmental Health, Institute of Computational Biology, Munich, Neuherberg, Germany
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Irmela Jeremias
1Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum München, German Center for Environmental Health (HMGU), 81377 Munich, Germany
2Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University of Munich (LMU), 81377 Munich, Germany
6German Consortium for Translational Cancer Research (DKTK), Partnering Site Munich, 80336 Munich, Germany
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Christian J Braun
2Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University of Munich (LMU), 81377 Munich, Germany
7Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technische Universität München, Munich, Germany
8Hopp Children’s Cancer Center Heidelberg (KiTZ), German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany
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  • For correspondence: chr.braun@med.lmu.de
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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 distinct 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.

Footnotes

  • ↵# Joint lead contact.

  • Wrong order of the author list.

  • http://www.crispr-clue.de

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 04, 2020.
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CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries
Martin Becker, Heidi Noll-Puchta, Diana Amend, Florian Nolte, Christiane Fuchs, Irmela Jeremias, Christian J Braun
bioRxiv 2020.04.02.021279; doi: https://doi.org/10.1101/2020.04.02.021279
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CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries
Martin Becker, Heidi Noll-Puchta, Diana Amend, Florian Nolte, Christiane Fuchs, Irmela Jeremias, Christian J Braun
bioRxiv 2020.04.02.021279; doi: https://doi.org/10.1101/2020.04.02.021279

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