Gene editing and scalable functional genomic screening in species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in , the causative agent of leishmaniasis. As lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in , , , and , 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 then generated a -optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in . 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 in via delivery of plasmid libraries.