Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments inLeishmania, the causative agent of leishmaniasis. AsLeishmanialack 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 multipleLeishmaniaspecies are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs inLeishmaniato introduce STOP codons by converting cytosine into thymine and created<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.leishbaseedit.net">www.leishbaseedit.net</ext-link>for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes inL. mexicana,L. major, L. donovaniandL. 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 then generated aLeishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen inL. 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 inLeishmaniavia delivery of plasmid libraries.