Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit
The recent adaptation of CRISPR Cas9 genome editing to Leishmania spp. has opened a new era in deciphering Leishmania biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion...
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doaj-ba51617605f94c0a97532594ed45ef772020-11-24T23:58:53ZengHindawi LimitedBioMed Research International2314-61332314-61412017-01-01201710.1155/2017/46356054635605Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 ToolkitDaniel Martel0Tom Beneke1Eva Gluenz2Gerald F. Späth3Najma Rachidi4Institut Pasteur and INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, Paris, FranceSir William Dunn School of Pathology, University of Oxford, Oxford, UKSir William Dunn School of Pathology, University of Oxford, Oxford, UKInstitut Pasteur and INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, Paris, FranceInstitut Pasteur and INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, Paris, FranceThe recent adaptation of CRISPR Cas9 genome editing to Leishmania spp. has opened a new era in deciphering Leishmania biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated in L. mexicana and L. major. In this study, we validated the toolkit in Leishmania donovani targeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit in L. donovani, which will be crucial for genetic modification of hamster-derived, disease-relevant parasites.http://dx.doi.org/10.1155/2017/4635605 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Daniel Martel Tom Beneke Eva Gluenz Gerald F. Späth Najma Rachidi |
spellingShingle |
Daniel Martel Tom Beneke Eva Gluenz Gerald F. Späth Najma Rachidi Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit BioMed Research International |
author_facet |
Daniel Martel Tom Beneke Eva Gluenz Gerald F. Späth Najma Rachidi |
author_sort |
Daniel Martel |
title |
Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit |
title_short |
Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit |
title_full |
Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit |
title_fullStr |
Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit |
title_full_unstemmed |
Characterisation of Casein Kinase 1.1 in Leishmania donovani Using the CRISPR Cas9 Toolkit |
title_sort |
characterisation of casein kinase 1.1 in leishmania donovani using the crispr cas9 toolkit |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2017-01-01 |
description |
The recent adaptation of CRISPR Cas9 genome editing to Leishmania spp. has opened a new era in deciphering Leishmania biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated in L. mexicana and L. major. In this study, we validated the toolkit in Leishmania donovani targeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit in L. donovani, which will be crucial for genetic modification of hamster-derived, disease-relevant parasites. |
url |
http://dx.doi.org/10.1155/2017/4635605 |
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