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Bar-seq strategies for the LeishGEdit toolbox

View ORCID ProfileTom Beneke, View ORCID ProfileEva Gluenz
doi: https://doi.org/10.1101/2020.03.19.998856
Tom Beneke
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
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  • For correspondence: tom.beneke@path.ox.ac.uk
Eva Gluenz
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
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  • ORCID record for Eva Gluenz
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Abstract

The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we developed previously a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit [1]. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations [2]. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligos are now accessible through our website www.leishgedit.net allowing to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.

Highlights

  • Developing tools for pooled bar-seq mutant screens across the kinetoplastid community

  • Development of a standalone script to design primers suitable for the LeishGEdit toolbox

  • Generation of 14,995 barcodes that can be used for bar-seq strategies in kinetoplastids

  • Bar-seq primers for all TriTrypDB genomes (release 41) can be obtained from www.leishgedit.net

Footnotes

  • http://www.leishgedit.net/

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 4.0 International license.
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Posted March 20, 2020.
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Bar-seq strategies for the LeishGEdit toolbox
Tom Beneke, Eva Gluenz
bioRxiv 2020.03.19.998856; doi: https://doi.org/10.1101/2020.03.19.998856
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Bar-seq strategies for the LeishGEdit toolbox
Tom Beneke, Eva Gluenz
bioRxiv 2020.03.19.998856; doi: https://doi.org/10.1101/2020.03.19.998856

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