CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining.
Biotechnol Bioeng
; 112(10): 2154-62, 2015 Oct.
Article
em En
| MEDLINE
| ID: mdl-25943095
Mammalian cells are widely used for the production of therapeutic recombinant proteins, as these cells facilitate accurate folding and post-translational modifications often essential for optimum activity. Targeted insertion of a plasmid harboring a gene of interest into the genome of mammalian cells for the expression of a desired protein is a key step in production of such biologics. Here we show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR-Cas9 system can be efficiently used to target â¼5 kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ). We were able to achieve efficiencies of up to 0.17% in HEK293 cells and 0.45% in CHO cells. This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in mammalian cells.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
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Recombinação Genética
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Engenharia Metabólica
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Sistemas CRISPR-Cas
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article