TY  - JOUR
A1  - Reuter, Christian
A1  - Hauf, Laura
A1  - Imdahl, Fabian
A1  - Sen, Rituparno
A1  - Vafadarnejad, Ehsan
A1  - Fey, Philipp
A1  - Finger, Tamara
A1  - Jones, Nicola G.
A1  - Walles, Heike
A1  - Barquist, Lars
A1  - Saliba, Antoine-Emmanuel
A1  - Groeber-Becker, Florian
A1  - Engstler, Markus
T1  - Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms
JF  - Nature Communications
N2  - Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.
KW  - mechanisms of disease
KW  - parasitology
KW  - transcriptomics
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-358142
VL  - 14
ER  - 
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  -