Bar-seq strategies for the LeishGEdit toolbox

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 previously developed 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 oligo sequences are now accessible through our website www.leishgedit.net allowing researchers 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.

Keywords: Bar-seq; CRISPR-Cas9; Gene editing; Kinetoplastids; LeishGEdit; Leishmania; Trypanosoma.