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

• 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
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep18341

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.