Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a.
Sci Rep
; 12(1): 17775, 2022 10 22.
Article
en En
| MEDLINE
| ID: mdl-36272994
ABSTRACT
The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sistemas CRISPR-Cas
/
Edición Génica
Límite:
Animals
Idioma:
En
Revista:
Sci Rep
Año:
2022
Tipo del documento:
Article
País de afiliación:
Japón