A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

OBJECTIVE: Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP)...

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Autores principales: Shaw, Sebastian, Knüsel, Sebastian, Hoenner, Sarah, Roditi, Isabel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Research Note
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268226/
https://www.ncbi.nlm.nih.gov/pubmed/32493474
http://dx.doi.org/10.1186/s13104-020-05089-z
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author Shaw, Sebastian
Knüsel, Sebastian
Hoenner, Sarah
Roditi, Isabel
author_facet Shaw, Sebastian
Knüsel, Sebastian
Hoenner, Sarah
Roditi, Isabel
author_sort Shaw, Sebastian
collection PubMed
description OBJECTIVE: Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. RESULTS: We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.
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spelling pubmed-72682262020-06-07 A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei Shaw, Sebastian Knüsel, Sebastian Hoenner, Sarah Roditi, Isabel BMC Res Notes Research Note OBJECTIVE: Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. RESULTS: We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes. BioMed Central 2020-06-03 /pmc/articles/PMC7268226/ /pubmed/32493474 http://dx.doi.org/10.1186/s13104-020-05089-z Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Note
Shaw, Sebastian
Knüsel, Sebastian
Hoenner, Sarah
Roditi, Isabel
A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title_full A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title_fullStr A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title_full_unstemmed A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title_short A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
title_sort transient crispr/cas9 expression system for genome editing in trypanosoma brucei
topic Research Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268226/
https://www.ncbi.nlm.nih.gov/pubmed/32493474
http://dx.doi.org/10.1186/s13104-020-05089-z
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