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

Abstract

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in <i>Leishmania</i>, the causative agent of leishmaniasis. As <i>Leishmania</i> 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 <i>Leishmania</i> 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 <i>Leishmania</i> 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 <i>L. mexicana</i>, <i>L. major</i>, <i>L. donovani</i>, and <i>L. infantum</i>, 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 <i>Leishmania</i>-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in <i>L. mexicana</i>. 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 <i>Leishmania</i> via delivery of plasmid libraries.