Abstract
Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.
Highlights
Genome editing techniques such as CRISPR/Cas[9] have both become common gene engineering technologies and have been applied to gene therapy
These results confirmed that adenovirus vector (AdV) is an excellent tool that can increase the efficiency of genome editing
These results show that Exci-Cas[9] is a short-term high expression system of Cas[9] controlled with high accuracy by Cre AdV (Cre) and with high efficiency of genome editing
Summary
Genome editing techniques such as CRISPR/Cas[9] have both become common gene engineering technologies and have been applied to gene therapy. The problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. The efficiencies of hepatitis B virus genome disruption by Exci-Cas[9] and Cas[9] expression by AdV directly (Avec) were the same, about 80–90%. We believe that Exci-Cas[9] developed in this study is useful for resolving the persistent expression of Cas[9], which has been a problem in genome editing, and for eliminating long-term DNA viruses such as human papilloma virus. Cas[9] nuclease and a guide RNA against a target DNA, are introduced into cells, site-specific double-stranded breaks (DSB) in the genome can be induced efficiently[5,6,7,8]. If AdV and a short-term expression system can be combined, the usefulness and safety of genome editing may be enhanced
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.