Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept.
CRISPR/Cas9
Entamoeba histolytica
Homologous recombination
Luciferase
Journal
International journal for parasitology
ISSN: 1879-0135
Titre abrégé: Int J Parasitol
Pays: England
ID NLM: 0314024
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
03
07
2020
revised:
05
09
2020
accepted:
10
09
2020
pubmed:
3
12
2020
medline:
12
10
2021
entrez:
2
12
2020
Statut:
ppublish
Résumé
The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.
Identifiants
pubmed: 33264648
pii: S0020-7519(20)30312-X
doi: 10.1016/j.ijpara.2020.09.005
pmc: PMC7880892
mid: NIHMS1652480
pii:
doi:
Substances chimiques
RNA, Guide
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
193-200Subventions
Organisme : NIGMS NIH HHS
ID : K12 GM088033
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI102277
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI149268
Pays : United States
Organisme : NIH HHS
ID : T35 OD010989
Pays : United States
Informations de copyright
Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.
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