TY  - JOUR
A1  - Engstler, Markus
A1  - Beneke, Tom
T1  - Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit
JF  - eLife
N2  - 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. mexicana, 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 Leishmania via delivery of plasmid libraries.
KW  - CRISPR/Cas9
KW  - Leishmania
KW  - cytosine base editor (CBE) toolbox
KW  - gene editing
KW  - scalable functional genomic screening
KW  - LeishBASEedit
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350002
VL  - 12
ER  -