Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit
Zitieren Sie bitte immer diese 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.…
Autor(en): | Markus Engstler, Tom BenekeORCiD |
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URN: | urn:nbn:de:bvb:20-opus-350002 |
Dokumentart: | Artikel / Aufsatz in einer Zeitschrift |
Institute der Universität: | Medizinische Fakultät / Theodor-Boveri-Institut für Biowissenschaften |
Sprache der Veröffentlichung: | Englisch |
Titel des übergeordneten Werkes / der Zeitschrift (Englisch): | eLife |
Erscheinungsjahr: | 2023 |
Band / Jahrgang: | 12 |
Aufsatznummer: | e85605 |
Originalveröffentlichung / Quelle: | eLife (2023) 12:e85605. DOI: 10.7554/eLife.85605 |
DOI: | https://doi.org/10.7554/eLife.85605 |
Allgemeine fachliche Zuordnung (DDC-Klassifikation): | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Freie Schlagwort(e): | CRISPR/Cas9; LeishBASEedit; Leishmania; cytosine base editor (CBE) toolbox; gene editing; scalable functional genomic screening |
Datum der Freischaltung: | 28.03.2024 |
Lizenz (Deutsch): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |