title: Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells.
abstract: 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.
authors.affiliation: Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Yoshioka
authors.affiliation: Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Fujii
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Sugiura
authors.affiliation: Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Tokyo 113-8657, Japan.
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Naito
mesh_terms: D064113:CRISPR-Cas Systems; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*
chemical_list: D012319:RNA Polymerase II; D012320:RNA Polymerase III