Abstract text
CRISPR Cas9 gene editing has revolutionized loss-of-function experiments in Leishmania, the causative agent of leishmanaisis. But since Leishmania lack a functional non-homologous DNA end joining pathway, obtaining null mutants typically requires the addition of donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. This makes genome-wide loss-of-function screens under different conditions and across multiple Leishmania species unfeasible to-date. Here, we report a CRISPR Cas9 Cytosine Base Editor (CBE) toolbox to overcome these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created www.leishbaseedit.net for CBE primer design in kinetoplastids. Through a series of reporter assays and by targeting single- und multi-copy genes in L. mexicana, L. major, L. donovani and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We also generated a Leishmania optimised CBE version and successfully targeted an essential gene in a small-scale plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA or isolation of clones, we believe that this enables for the first time functional genetic screens across multiple Leishmania species.