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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Bibliographic Details
Published in:BioMed research international 2017-01, Vol.2017 (2017), p.1-11
Main Authors: Späth, Gerald F., Gluenz, Eva, Beneke, Tom, Martel, Daniel, Rachidi, Najma
Format: Article
Language:English
Subjects:
Amastigotes
Animals
Casein
Casein kinase I
Casein Kinase I/genetics
Clonal deletion
Cloning
Cricetinae
CRISPR
CRISPR-Cas Systems/genetics
DNA sequencing
Flagella
Gene Deletion
Gene Editing
Genetic aspects
Genetic engineering
Genetic modification
Genome editing
Genomes
Health aspects
Humans
Kinases
Leishmania
Leishmania donovani
Leishmania donovani/genetics
Leishmania donovani/pathogenicity
Leishmaniasis, Visceral/genetics
Leishmaniasis, Visceral/therapy
Life Sciences
Methods
Microbiology and Parasitology
Mutants
Nucleotide sequencing
Parasites
Parasitic diseases
Phosphotransferases
Promastigotes
Protozoa
Protozoan Proteins/genetics
Stationary phase
Trypanosoma brucei
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Summary: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.
ISSN:2314-6133
2314-6141
DOI:10.1155/2017/4635605