CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout.
MethodsX
; 13: 102817, 2024 Dec.
Article
em En
| MEDLINE
| ID: mdl-39049926
ABSTRACT
Aedes mosquitoes are important virus vectors. We provide a toolkit for CRISPR-Cas9-editing of difficult-to-knockdown gene previously shown to be refractory to siRNA silencing in mosquito cells, which is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations. Starting from database searches of Ae. albopictus and the C6/36 cell line whole genome shotgun sequences for the prohibitin 2 (PHB2) gene, primers were designed to confirm the gene sequence in our laboratory-passaged C6/36 cell line for the correct design and cloning of CRISPR RNA into an insect plasmid vector to create a single guide RNA for the PHB2 gene target. After transfection of this plasmid vector into the C6/36 cells, cell clones selected by puromycin and/or limiting dilution were analyzed for insertions and deletions (INDELs) using PCR, sequencing and computational sequence decomposition. From this, we have identified mono-allelic and bi-allelic knockout cell clones. Using a mono-allelic knockout cell clone as an example, we characterized its INDELs by molecular cloning and computational analysis. Importantly, mono-allelic knockout was sufficient to reduce >80 % of PHB2 expression, which led to phenotypic switching and the propensity to form foci but was insufficient to affect growth rate or to inhibit Zika virus infection.â¢We provide a toolkit for CRISPR-Cas9-editing of the virus vector, Aedes albopictus C6/36 cell lineâ¢We validate this using a difficult-to-knockdown gene prohibitin 2â¢This toolkit is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations.
Texto completo:
1
Base de dados:
MEDLINE
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article