Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome
- Goethe University Frankfurt, Germany
- University of California, San Diego, United States
Abstract
Current technologies to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology to efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents that uncouples sequence diversity from sequence distribution. We demonstrate fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed at generating the up-to-date largest gRNA library to simultaneously interrogate the coding and noncoding human genome and identify genes, predicted promoter flanking regions, transcription factor and CTCF binding sites linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Data availability
All data generated or analysed during this study are included in the manuscript, supplementary files or are available through GitHub or Dryad. NGS data and custom software is available as supplementary files and from Dryad and GitHub. Plasmids encoding oTGW 3Cs-gRNA libraries will be made available through the Goethe University Depository (http://innovectis.de/technologien/goethe-depository/).
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Data from: Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genomeDryad Digital Repository, doi:10.5061/dryad.rs432pr.
Article and author information
Author details
Funding
Hessisches Ministerium für Wissenschaft und Kunst (IIIL5-518/17.004)
- Manuel Kaulich
Deutsche Forschungsgemeinschaft (EXC115/2)
- Manuel Kaulich
Hessisches Ministerium für Wissenschaft und Kunst (IIIL5-519/03/03.001)
- Manuel Kaulich
Deutsche Forschungsgemeinschaft (EXC147/2)
- Manuel Kaulich
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jonathan S Weissman, University of California, San Francisco, United States
Version history
- Received: October 7, 2018
- Accepted: February 25, 2019
- Accepted Manuscript published: March 6, 2019 (version 1)
- Version of Record published: March 19, 2019 (version 2)
Copyright
© 2019, Wegner et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome
eLife 8:e42549.
https://doi.org/10.7554/eLife.42549
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