A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9.
Mol Ther Methods Clin Dev
; 2: 15035, 2015.
Article
de En
| MEDLINE
| ID: mdl-26417609
ABSTRACT
The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Langue:
En
Journal:
Mol Ther Methods Clin Dev
Année:
2015
Type de document:
Article
Pays d'affiliation:
Royaume-Uni