Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells

The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a...

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Bibliographic Details
Published in:Scientific reports 2015-12, Vol.5 (1), p.18341-18341, Article 18341
Main Authors: Yoshioka, Shin, Fujii, Wataru, Ogawa, Tetsuhiro, Sugiura, Koji, Naito, Kunihiko
Format: Article
Language:English
Subjects:
631/1647/1511
631/337/1427/2122
CRISPR
CRISPR-Cas Systems
DNA-directed RNA polymerase
Gene Expression
gRNA
HEK293 Cells
Humanities and Social Sciences
Humans
Mammalian cells
mRNA
multidisciplinary
Plasmids
Promoter Regions, Genetic
RNA polymerase
RNA Polymerase II - metabolism
RNA Polymerase III - metabolism
Science
Transfection
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Summary:The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep18341