Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Please always quote using this URN: urn:nbn:de:bvb:20-opus-350002
  • 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 editorCRISPR/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.show moreshow less

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Metadaten
Author: Markus Engstler, Tom BenekeORCiD
URN:urn:nbn:de:bvb:20-opus-350002
Document Type:Journal article
Faculties:Medizinische Fakultät / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):eLife
Year of Completion:2023
Volume:12
Article Number:e85605
Source:eLife (2023) 12:e85605. DOI: 10.7554/eLife.85605
DOI:https://doi.org/10.7554/eLife.85605
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Tag:CRISPR/Cas9; LeishBASEedit; Leishmania; cytosine base editor (CBE) toolbox; gene editing; scalable functional genomic screening
Release Date:2024/03/28
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International