Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.
J Gene Med
; 21(11): e3115, 2019 11.
Article
em En
| MEDLINE
| ID: mdl-31348845
ABSTRACT
BACKGROUND:
Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.METHODS:
Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.RESULTS:
We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.CONCLUSIONS:
We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Cosmídeos
/
Expressão Gênica
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Amplificação de Genes
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RNA Guia de Cinetoplastídeos
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Sistemas CRISPR-Cas
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Edição de Genes
Limite:
Humans
Idioma:
En
Revista:
J Gene Med
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA MEDICA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Japão