Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit
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-a...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-05-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/85605 |
| _version_ | 1797821226007134208 |
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| author | Markus Engstler Tom Beneke |
| author_facet | Markus Engstler Tom Beneke |
| author_sort | Markus Engstler |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T09:49:41Z |
| format | Article |
| id | doaj.art-8a639d6117d649acac60022e7cdb3cf6 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-13T09:49:41Z |
| publishDate | 2023-05-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-8a639d6117d649acac60022e7cdb3cf62023-05-24T14:29:12ZengeLife Sciences Publications LtdeLife2050-084X2023-05-011210.7554/eLife.85605Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEeditMarkus Engstler0Tom Beneke1https://orcid.org/0000-0001-9117-2649Department of Cell and Developmental Biology, Biocentre, University of Würzburg, Wuerzburg, GermanyDepartment of Cell and Developmental Biology, Biocentre, University of Würzburg, Wuerzburg, GermanyCRISPR/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.https://elifesciences.org/articles/85605LeishmaniaCRISPR/Cas9cytosine base editorloss-of-function screeningLeishBASEedit |
| spellingShingle | Markus Engstler Tom Beneke Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit eLife Leishmania CRISPR/Cas9 cytosine base editor loss-of-function screening LeishBASEedit |
| title | Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit |
| title_full | Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit |
| title_fullStr | Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit |
| title_full_unstemmed | Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit |
| title_short | Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit |
| title_sort | gene editing and scalable functional genomic screening in leishmania species using the crispr cas9 cytosine base editor toolbox leishbaseedit |
| topic | Leishmania CRISPR/Cas9 cytosine base editor loss-of-function screening LeishBASEedit |
| url | https://elifesciences.org/articles/85605 |
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