Mar 20, 2020

Bar-seq strategies for the LeishGEdit toolbox

bioRxiv
Tom Beneke, Eva Gluenz

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. 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. 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.

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Mentioned in this Paper

Gene Editing
DNA Barcode, Taxonomic
Gene Function
Genes
Clustered Regularly Interspaced Short Palindromic Repeats
Nucleotides
Genome
Tissue Dissection
Analysis
DNA Barcoding, Taxonomic

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