Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.
CRISPR/Cas9
efficiency
kinetoplastids
marker‐free
protist
ribonucleoprotein complex transfection
universal
Journal
Molecular microbiology
ISSN: 1365-2958
Titre abrégé: Mol Microbiol
Pays: England
ID NLM: 8712028
Informations de publication
Date de publication:
01 Apr 2024
01 Apr 2024
Historique:
revised:
13
02
2024
received:
27
10
2023
accepted:
14
03
2024
medline:
1
4
2024
pubmed:
1
4
2024
entrez:
1
4
2024
Statut:
aheadofprint
Résumé
Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : CIHR
Pays : Canada
Informations de copyright
© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
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