Journal
ELIFE
Volume 12, Issue -, Pages -Publisher
eLIFE SCIENCES PUBL LTD
DOI: 10.7554/eLife.85605
Keywords
Leishmania???????; CRISPR/Cas9; cytosine base editor; loss-of-function screening; LeishBASEedit
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CRISPR/Cas9 gene editing has revolutionized loss-of-function experiments in Leishmania by introducing STOP codons and generating functional null mutants. This study provides a toolbox for CRISPR/Cas9 cytosine base editing in Leishmania, enabling efficient editing and facilitating functional genetic screens in non-clonal populations. This method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones.
CRISPR/Cas9 gene editing has revolutionised loss- of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania 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 Leishmania 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 Leishmania 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 L. mexi-cana, 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 then generated a Leishmania- optimised CBE and successfully targeted an essential gene in a 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 in Leish-mania via delivery of plasmid libraries.
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