Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template.
Sci Rep
; 11(1): 18501, 2021 09 16.
Article
en En
| MEDLINE
| ID: mdl-34531479
The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plasmodium falciparum
/
Sistemas CRISPR-Cas
/
Proteína 9 Asociada a CRISPR
Límite:
Animals
Idioma:
En
Revista:
Sci Rep
Año:
2021
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Reino Unido