Author:
Beneke Tom,Engstler Markus
Abstract
ABSTRACTCRISPR/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 createdwww.leishbaseedit.netfor 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.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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