Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.
CRISPR-Cas9
CreLox
genome edition
inducible knockout
parasite
trypanosomatids
Journal
Cellular microbiology
ISSN: 1462-5822
Titre abrégé: Cell Microbiol
Pays: India
ID NLM: 100883691
Informations de publication
Date de publication:
05 2020
05 2020
Historique:
received:
20
11
2019
revised:
24
12
2019
accepted:
27
12
2019
pubmed:
8
1
2020
medline:
3
6
2021
entrez:
8
1
2020
Statut:
ppublish
Résumé
Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.
Substances chimiques
Proto-Oncogene Proteins c-crk
0
Cre recombinase
EC 2.7.7.-
Integrases
EC 2.7.7.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e13159Informations de copyright
© 2020 John Wiley & Sons Ltd.
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