{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,4,11]],"date-time":"2025-04-11T05:30:23Z","timestamp":1744349423602},"reference-count":62,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,12,21]],"date-time":"2021-12-21T00:00:00Z","timestamp":1640044800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,12,21]],"date-time":"2021-12-21T00:00:00Z","timestamp":1640044800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"the Research Program on Hepatitis from the Japan Agency for Medical research and Development","award":["Grant Number JP20fk0310102"],"award-info":[{"award-number":["Grant Number JP20fk0310102"]}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>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\u00a0months. 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\u201390%. 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.<\/jats:p>","DOI":"10.1038\/s41598-021-03803-w","type":"journal-article","created":{"date-parts":[[2021,12,21]],"date-time":"2021-12-21T11:03:53Z","timestamp":1640084633000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre"],"prefix":"10.1038","volume":"11","author":[{"given":"Sayaka","family":"Nagamoto","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miyuki","family":"Agawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emi","family":"Tsuchitani","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kazunori","family":"Akimoto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saki Kondo","family":"Matsushima","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yumi","family":"Kanegae","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2021,12,21]]},"reference":[{"key":"3803_CR1","doi-asserted-by":"publisher","first-page":"1262","DOI":"10.1016\/j.cell.2014.05.010","volume":"157","author":"PD Hsu","year":"2014","unstructured":"Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262\u20131278. https:\/\/doi.org\/10.1016\/j.cell.2014.05.010 (2014).","journal-title":"Cell"},{"key":"3803_CR2","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1038\/s41586-020-1978-5","volume":"578","author":"JA Doudna","year":"2020","unstructured":"Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229\u2013236. https:\/\/doi.org\/10.1038\/s41586-020-1978-5 (2020).","journal-title":"Nature"},{"key":"3803_CR3","doi-asserted-by":"publisher","first-page":"1565","DOI":"10.1046\/j.1365-2958.2002.02839.x","volume":"43","author":"R Jansen","year":"2002","unstructured":"Jansen, R., Embden, J. D., Gaastra, W. & Schouls, L. M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565\u20131575. https:\/\/doi.org\/10.1046\/j.1365-2958.2002.02839.x (2002).","journal-title":"Mol. Microbiol."},{"key":"3803_CR4","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1126\/science.1179555","volume":"327","author":"P Horvath","year":"2010","unstructured":"Horvath, P. & Barrangou, R. CRISPR\/Cas, the immune system of bacteria and archaea. Science 327, 167\u2013170. https:\/\/doi.org\/10.1126\/science.1179555 (2010).","journal-title":"Science"},{"key":"3803_CR5","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1126\/science.1225829","volume":"337","author":"M Jinek","year":"2012","unstructured":"Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816\u2013821. https:\/\/doi.org\/10.1126\/science.1225829 (2012).","journal-title":"Science"},{"key":"3803_CR6","doi-asserted-by":"publisher","first-page":"1002","DOI":"10.1038\/nbt.2355","volume":"30","author":"L Qi","year":"2012","unstructured":"Qi, L., Haurwitz, R. E., Shao, W., Doudna, J. A. & Arkin, A. P. RNA processing enables predictable programming of gene expression. Nat. Biotechnol. 30, 1002\u20131006. https:\/\/doi.org\/10.1038\/nbt.2355 (2012).","journal-title":"Nat. Biotechnol."},{"key":"3803_CR7","doi-asserted-by":"publisher","first-page":"1380","DOI":"10.1016\/j.cell.2013.08.021","volume":"154","author":"FA Ran","year":"2013","unstructured":"Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380\u20131389. https:\/\/doi.org\/10.1016\/j.cell.2013.08.021 (2013).","journal-title":"Cell"},{"key":"3803_CR8","doi-asserted-by":"publisher","first-page":"2281","DOI":"10.1038\/nprot.2013.143","volume":"8","author":"FA Ran","year":"2013","unstructured":"Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281\u20132308. https:\/\/doi.org\/10.1038\/nprot.2013.143 (2013).","journal-title":"Nat. Protoc."},{"key":"3803_CR9","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1016\/j.scr.2017.12.015","volume":"27","author":"CW Chang","year":"2018","unstructured":"Chang, C. W., Chang, C. C., Hsia, K. C. & Tsai, S. Y. Generation of FHL2 homozygous knockout lines from human embryonic stem cells by CRISPR\/Cas9-mediated ablation. Stem Cell Res. 27, 21\u201324. https:\/\/doi.org\/10.1016\/j.scr.2017.12.015 (2018).","journal-title":"Stem Cell Res."},{"key":"3803_CR10","doi-asserted-by":"publisher","first-page":"910","DOI":"10.1016\/j.cell.2013.04.025","volume":"153","author":"H Wang","year":"2013","unstructured":"Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR\/Cas-mediated genome engineering. Cell 153, 910\u2013918. https:\/\/doi.org\/10.1016\/j.cell.2013.04.025 (2013).","journal-title":"Cell"},{"key":"3803_CR11","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw064","volume":"44","author":"X He","year":"2016","unstructured":"He, X. et al. Knock-in of large reporter genes in human cells via CRISPR\/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res. 44, e85. https:\/\/doi.org\/10.1093\/nar\/gkw064 (2016).","journal-title":"Nucleic Acids Res."},{"key":"3803_CR12","doi-asserted-by":"publisher","first-page":"E10745","DOI":"10.1073\/pnas.1711979114","volume":"114","author":"A Paix","year":"2017","unstructured":"Paix, A. et al. Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. Proc. Natl. Acad. Sci. U.S.A. 114, E10745\u2013E10754. https:\/\/doi.org\/10.1073\/pnas.1711979114 (2017).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"3803_CR13","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1038\/nature14299","volume":"520","author":"FA Ran","year":"2015","unstructured":"Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186\u2013191. https:\/\/doi.org\/10.1038\/nature14299 (2015).","journal-title":"Nature"},{"key":"3803_CR14","doi-asserted-by":"publisher","first-page":"1168","DOI":"10.1016\/j.ymthe.2017.03.012","volume":"25","author":"C Yin","year":"2017","unstructured":"Yin, C. et al. In vivo excision of HIV-1 provirus by saCas9 and multiplex single-guide RNAs in animal models. Mol. Ther. 25, 1168\u20131186. https:\/\/doi.org\/10.1016\/j.ymthe.2017.03.012 (2017).","journal-title":"Mol. Ther."},{"key":"3803_CR15","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1038\/nbt.2800","volume":"32","author":"H Koike-Yusa","year":"2014","unstructured":"Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267\u2013273. https:\/\/doi.org\/10.1038\/nbt.2800 (2014).","journal-title":"Nat. Biotechnol."},{"key":"3803_CR16","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku749","volume":"42","author":"AM Kabadi","year":"2014","unstructured":"Kabadi, A. M., Ousterout, D. G., Hilton, I. B. & Gersbach, C. A. Multiplex CRISPR\/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. 42, e147. https:\/\/doi.org\/10.1093\/nar\/gku749 (2014).","journal-title":"Nucleic Acids Res."},{"key":"3803_CR17","doi-asserted-by":"publisher","first-page":"2153","DOI":"10.12688\/f1000research.11243.1","volume":"6","author":"CH Lau","year":"2017","unstructured":"Lau, C. H. & Suh, Y. In vivo genome editing in animals using AAV-CRISPR system: Applications to translational research of human disease. F1000Res. 6, 2153. https:\/\/doi.org\/10.12688\/f1000research.11243.1 (2017).","journal-title":"F1000Res."},{"key":"3803_CR18","doi-asserted-by":"publisher","first-page":"328","DOI":"10.1038\/nbt.3471","volume":"34","author":"H Yin","year":"2016","unstructured":"Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328\u2013333. https:\/\/doi.org\/10.1038\/nbt.3471 (2016).","journal-title":"Nat. Biotechnol."},{"key":"3803_CR19","doi-asserted-by":"publisher","first-page":"5105","DOI":"10.1038\/srep05105","volume":"4","author":"I Maggio","year":"2014","unstructured":"Maggio, I. et al. Adenoviral vector delivery of RNA-guided CRISPR\/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci. Rep. 4, 5105. https:\/\/doi.org\/10.1038\/srep05105 (2014).","journal-title":"Sci. Rep."},{"key":"3803_CR20","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1089\/hum.2015.087","volume":"26","author":"D Wang","year":"2015","unstructured":"Wang, D. et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR\/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432\u2013442. https:\/\/doi.org\/10.1089\/hum.2015.087 (2015).","journal-title":"Hum. Gene Ther."},{"key":"3803_CR21","doi-asserted-by":"publisher","first-page":"488","DOI":"10.1161\/CIRCRESAHA.115.304351","volume":"115","author":"Q Ding","year":"2014","unstructured":"Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488\u2013492. https:\/\/doi.org\/10.1161\/CIRCRESAHA.115.304351 (2014).","journal-title":"Circ. Res."},{"key":"3803_CR22","doi-asserted-by":"publisher","first-page":"3954","DOI":"10.1016\/j.febslet.2014.09.008","volume":"588","author":"R Cheng","year":"2014","unstructured":"Cheng, R. et al. Efficient gene editing in adult mouse livers via adenoviral delivery of CRISPR\/Cas9. FEBS Lett. 588, 3954\u20133958. https:\/\/doi.org\/10.1016\/j.febslet.2014.09.008 (2014).","journal-title":"FEBS Lett."},{"key":"3803_CR23","doi-asserted-by":"publisher","first-page":"3961","DOI":"10.1038\/s41598-021-83259-0","volume":"11","author":"T Nakanishi","year":"2021","unstructured":"Nakanishi, T. et al. Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR\/Cas9 and in vivo genome editing. Sci. Rep. 11, 3961. https:\/\/doi.org\/10.1038\/s41598-021-83259-0 (2021).","journal-title":"Sci. Rep."},{"key":"3803_CR24","doi-asserted-by":"publisher","first-page":"925","DOI":"10.1089\/hum.2007.085","volume":"18","author":"M Nakai","year":"2007","unstructured":"Nakai, M. et al. Expression of pIX gene induced by transgene promoter: Possible cause of host immune response in first-generation adenoviral vectors. Hum. Gene Ther. 18, 925\u2013936. https:\/\/doi.org\/10.1089\/hum.2007.085 (2007).","journal-title":"Hum. Gene Ther."},{"key":"3803_CR25","doi-asserted-by":"publisher","first-page":"1118","DOI":"10.1056\/NEJMra031087","volume":"350","author":"D Ganem","year":"2004","unstructured":"Ganem, D. & Prince, A. M. Hepatitis B virus infection\u2014Natural history and clinical consequences. N. Engl. J. Med. 350, 1118\u20131129. https:\/\/doi.org\/10.1056\/NEJMra031087 (2004).","journal-title":"N. Engl. J. Med."},{"key":"3803_CR26","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1046\/j.1365-2893.2003.00487.x","volume":"11","author":"D Lavanchy","year":"2004","unstructured":"Lavanchy, D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J. Viral Hepat. 11, 97\u2013107. https:\/\/doi.org\/10.1046\/j.1365-2893.2003.00487.x (2004).","journal-title":"J. Viral Hepat."},{"key":"3803_CR27","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1016\/S0140-6736(19)32989-7","volume":"395","author":"KE Rudd","year":"2020","unstructured":"Rudd, K. E. et al. Global, regional, and national sepsis incidence and mortality, 1990\u20132017: Analysis for the Global Burden of Disease Study. Lancet 395, 200\u2013211. https:\/\/doi.org\/10.1016\/S0140-6736(19)32989-7 (2020).","journal-title":"Lancet"},{"key":"3803_CR28","doi-asserted-by":"publisher","first-page":"672","DOI":"10.1016\/j.virol.2015.02.031","volume":"479\u2013480","author":"C Seeger","year":"2015","unstructured":"Seeger, C. & Mason, W. S. Molecular biology of hepatitis B virus infection. Virology 479\u2013480, 672\u2013686. https:\/\/doi.org\/10.1016\/j.virol.2015.02.031 (2015).","journal-title":"Virology"},{"key":"3803_CR29","doi-asserted-by":"publisher","first-page":"10833","DOI":"10.1038\/srep10833","volume":"5","author":"V Ramanan","year":"2015","unstructured":"Ramanan, V. et al. CRISPR\/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci. Rep. 5, 10833. https:\/\/doi.org\/10.1038\/srep10833 (2015).","journal-title":"Sci. Rep."},{"key":"3803_CR30","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1016\/j.antiviral.2015.03.015","volume":"118","author":"C Dong","year":"2015","unstructured":"Dong, C. et al. Targeting hepatitis B virus cccDNA by CRISPR\/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res. 118, 110\u2013117. https:\/\/doi.org\/10.1016\/j.antiviral.2015.03.015 (2015).","journal-title":"Antiviral Res."},{"key":"3803_CR31","doi-asserted-by":"publisher","first-page":"13734","DOI":"10.1038\/srep13734","volume":"5","author":"M Karimova","year":"2015","unstructured":"Karimova, M. et al. CRISPR\/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci. Rep. 5, 13734. https:\/\/doi.org\/10.1038\/srep13734 (2015).","journal-title":"Sci. Rep."},{"key":"3803_CR32","doi-asserted-by":"publisher","first-page":"1258","DOI":"10.1038\/mt.2016.94","volume":"24","author":"C Seeger","year":"2016","unstructured":"Seeger, C. & Sohn, J. A. Complete spectrum of CRISPR\/Cas9-induced mutations on HBV cccDNA. Mol. Ther. 24, 1258\u20131266. https:\/\/doi.org\/10.1038\/mt.2016.94 (2016).","journal-title":"Mol. Ther."},{"key":"3803_CR33","doi-asserted-by":"publisher","first-page":"6122","DOI":"10.1038\/s41598-017-05905-w","volume":"7","author":"T Kurihara","year":"2017","unstructured":"Kurihara, T. et al. Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9. Sci. Rep. 7, 6122. https:\/\/doi.org\/10.1038\/s41598-017-05905-w (2017).","journal-title":"Sci. Rep."},{"key":"3803_CR34","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1016\/j.virol.2014.12.001","volume":"476","author":"EM Kennedy","year":"2015","unstructured":"Kennedy, E. M. et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR\/Cas RNA-guided DNA endonuclease. Virology 476, 196\u2013205. https:\/\/doi.org\/10.1016\/j.virol.2014.12.001 (2015).","journal-title":"Virology"},{"key":"3803_CR35","doi-asserted-by":"publisher","first-page":"188","DOI":"10.1016\/j.antiviral.2015.10.004","volume":"123","author":"EM Kennedy","year":"2015","unstructured":"Kennedy, E. M., Kornepati, A. V. & Cullen, B. R. Targeting hepatitis B virus cccDNA using CRISPR\/Cas9. Antiviral Res. 123, 188\u2013192. https:\/\/doi.org\/10.1016\/j.antiviral.2015.10.004 (2015).","journal-title":"Antiviral Res."},{"key":"3803_CR36","doi-asserted-by":"publisher","first-page":"30675","DOI":"10.18632\/oncotarget.5169","volume":"6","author":"S Chira","year":"2015","unstructured":"Chira, S. et al. Progresses towards safe and efficient gene therapy vectors. Oncotarget 6, 30675\u201330703. https:\/\/doi.org\/10.18632\/oncotarget.5169 (2015).","journal-title":"Oncotarget"},{"key":"3803_CR37","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkq966","volume":"39","author":"Y Kanegae","year":"2011","unstructured":"Kanegae, Y. et al. High-level expression by tissue\/cancer-specific promoter with strict specificity using a single-adenoviral vector. Nucleic Acids Res. 39, e7. https:\/\/doi.org\/10.1093\/nar\/gkq966 (2011).","journal-title":"Nucleic Acids Res."},{"key":"3803_CR38","doi-asserted-by":"publisher","first-page":"41851","DOI":"10.1038\/srep41851","volume":"7","author":"M Suzuki","year":"2017","unstructured":"Suzuki, M. et al. Efficient genome replication of hepatitis B virus using adenovirus vector: A compact pregenomic RNA-expression unit. Sci. Rep. 7, 41851. https:\/\/doi.org\/10.1038\/srep41851 (2017).","journal-title":"Sci. Rep."},{"key":"3803_CR39","doi-asserted-by":"publisher","first-page":"466","DOI":"10.1128\/mcb.8.1.466-472.1988","volume":"8","author":"Y Takebe","year":"1988","unstructured":"Takebe, Y. et al. SR alpha promoter: An efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8, 466\u2013472. https:\/\/doi.org\/10.1128\/mcb.8.1.466-472.1988 (1988).","journal-title":"Mol. Cell. Biol."},{"key":"3803_CR40","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1038\/nbt.3081","volume":"33","author":"JA Zuris","year":"2015","unstructured":"Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73\u201380. https:\/\/doi.org\/10.1038\/nbt.3081 (2015).","journal-title":"Nat. Biotechnol."},{"key":"3803_CR41","doi-asserted-by":"publisher","first-page":"9987","DOI":"10.1128\/JVI.00751-10","volume":"84","author":"SL Stephen","year":"2010","unstructured":"Stephen, S. L. et al. Chromosomal integration of adenoviral vector DNA in vivo. J. Virol. 84, 9987\u20139994. https:\/\/doi.org\/10.1128\/JVI.00751-10 (2010).","journal-title":"J. Virol."},{"key":"3803_CR42","doi-asserted-by":"publisher","first-page":"1645","DOI":"10.1073\/pnas.94.5.1645","volume":"94","author":"HH Chen","year":"1997","unstructured":"Chen, H. H. et al. Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. U.S.A. 94, 1645\u20131650. https:\/\/doi.org\/10.1073\/pnas.94.5.1645 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"3803_CR43","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1007\/s00439-016-1686-2","volume":"135","author":"D Stone","year":"2016","unstructured":"Stone, D., Niyonzima, N. & Jerome, K. R. Genome editing and the next generation of antiviral therapy. Hum. Genet. 135, 1071\u20131082. https:\/\/doi.org\/10.1007\/s00439-016-1686-2 (2016).","journal-title":"Hum. Genet."},{"key":"3803_CR44","doi-asserted-by":"publisher","first-page":"204993612096502","DOI":"10.1177\/2049936120965027","volume":"7","author":"V Soriano","year":"2020","unstructured":"Soriano, V. et al. Advances in hepatitis B therapeutics. Ther. Adv. Infect. Dis. 7, 2049936120965027. https:\/\/doi.org\/10.1177\/2049936120965027 (2020).","journal-title":"Ther. Adv. Infect. Dis."},{"key":"3803_CR45","doi-asserted-by":"publisher","first-page":"258","DOI":"10.1016\/j.omtm.2020.11.014","volume":"20","author":"D Stone","year":"2021","unstructured":"Stone, D. et al. CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice. Mol. Ther. Methods Clin. Dev. 20, 258\u2013275. https:\/\/doi.org\/10.1016\/j.omtm.2020.11.014 (2021).","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"3803_CR46","doi-asserted-by":"publisher","first-page":"636","DOI":"10.1016\/j.jhep.2006.05.019","volume":"45","author":"D Sun","year":"2006","unstructured":"Sun, D. & Nassal, M. Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus. J. Hepatol. 45, 636\u2013645. https:\/\/doi.org\/10.1016\/j.jhep.2006.05.019 (2006).","journal-title":"J. Hepatol."},{"key":"3803_CR47","doi-asserted-by":"publisher","first-page":"2836","DOI":"10.1128\/JVI.62.8.2836-2844.1988","volume":"62","author":"MA Sells","year":"1988","unstructured":"Sells, M. A., Zelent, A. Z., Shvartsman, M. & Acs, G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J. Virol. 62, 2836\u20132844. https:\/\/doi.org\/10.1128\/JVI.62.8.2836-2844.1988 (1988).","journal-title":"J. Virol."},{"key":"3803_CR48","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1016\/0378-1119(90)90091-5","volume":"91","author":"DW Kim","year":"1990","unstructured":"Kim, D. W., Uetsuki, T., Kaziro, Y., Yamaguchi, N. & Sugano, S. Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene 91, 217\u2013223. https:\/\/doi.org\/10.1016\/0378-1119(90)90091-5 (1990).","journal-title":"Gene"},{"key":"3803_CR49","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1016\/j.jhep.2006.03.014","volume":"45","author":"A Parouchev","year":"2006","unstructured":"Parouchev, A. et al. Efficient ex vivo gene transfer into non-human primate hepatocytes using HIV-1 derived lentiviral vectors. J. Hepatol. 45, 99\u2013107. https:\/\/doi.org\/10.1016\/j.jhep.2006.03.014 (2006).","journal-title":"J. Hepatol."},{"key":"3803_CR50","doi-asserted-by":"publisher","first-page":"3816","DOI":"10.1093\/nar\/23.19.3816","volume":"23","author":"Y Kanegae","year":"1995","unstructured":"Kanegae, Y. et al. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res. 23, 3816\u20133821. https:\/\/doi.org\/10.1093\/nar\/23.19.3816 (1995).","journal-title":"Nucleic Acids Res."},{"key":"3803_CR51","doi-asserted-by":"publisher","first-page":"430","DOI":"10.1038\/mt.2016.10","volume":"24","author":"ML Maeder","year":"2016","unstructured":"Maeder, M. L. & Gersbach, C. A. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430\u2013446. https:\/\/doi.org\/10.1038\/mt.2016.10 (2016).","journal-title":"Mol. Ther."},{"key":"3803_CR52","doi-asserted-by":"publisher","first-page":"564","DOI":"10.1038\/sj.gt.3300861","volume":"6","author":"SI Miyatake","year":"1999","unstructured":"Miyatake, S. I. et al. Hepatoma-specific antitumor activity of an albumin enhancer\/promoter regulated herpes simplex virus in vivo. Gene Ther. 6, 564\u2013572. https:\/\/doi.org\/10.1038\/sj.gt.3300861 (1999).","journal-title":"Gene Ther."},{"key":"3803_CR53","doi-asserted-by":"publisher","first-page":"819","DOI":"10.1126\/science.1231143","volume":"339","author":"L Cong","year":"2013","unstructured":"Cong, L. et al. Multiplex genome engineering using CRISPR\/Cas systems. Science 339, 819\u2013823. https:\/\/doi.org\/10.1126\/science.1231143 (2013).","journal-title":"Science"},{"key":"3803_CR54","doi-asserted-by":"publisher","first-page":"1143","DOI":"10.1089\/hum.2010.245","volume":"22","author":"SJ Gray","year":"2011","unstructured":"Gray, S. J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 22, 1143\u20131153. https:\/\/doi.org\/10.1089\/hum.2010.245 (2011).","journal-title":"Hum. Gene Ther."},{"key":"3803_CR55","doi-asserted-by":"publisher","first-page":"1136","DOI":"10.1038\/srep01136","volume":"3","author":"A Maekawa","year":"2013","unstructured":"Maekawa, A. et al. Efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery. Sci. Rep. 3, 1136. https:\/\/doi.org\/10.1038\/srep01136 (2013).","journal-title":"Sci. Rep."},{"key":"3803_CR56","doi-asserted-by":"publisher","DOI":"10.1002\/jgm.3115","volume":"21","author":"T Nakanishi","year":"2019","unstructured":"Nakanishi, T. et al. Highly multiplex guide RNA expression units of CRISPR\/Cas9 were completely stable using cosmid amplification in a novel polygonal structure. J. Gene Med. 21, e3115. https:\/\/doi.org\/10.1002\/jgm.3115 (2019).","journal-title":"J. Gene Med."},{"key":"3803_CR57","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1038\/gt.2014.124","volume":"22","author":"M Suzuki","year":"2015","unstructured":"Suzuki, M. et al. Preferable sites and orientations of transgene inserted in the adenovirus vector genome: The E3 site may be unfavorable for transgene position. Gene Ther. 22, 421\u2013429. https:\/\/doi.org\/10.1038\/gt.2014.124 (2015).","journal-title":"Gene Ther."},{"key":"3803_CR58","doi-asserted-by":"publisher","first-page":"157","DOI":"10.7883\/yoken1952.47.157","volume":"47","author":"Y Kanegae","year":"1994","unstructured":"Kanegae, Y., Makimura, M. & Saito, I. A simple and efficient method for purification of infectious recombinant adenovirus. Jpn. J. Med. Sci. Biol. 47, 157\u2013166. https:\/\/doi.org\/10.7883\/yoken1952.47.157 (1994).","journal-title":"Jpn. J. Med. Sci. Biol."},{"key":"3803_CR59","doi-asserted-by":"publisher","first-page":"945","DOI":"10.1016\/j.bbrc.2011.12.016","volume":"417","author":"Z Pei","year":"2012","unstructured":"Pei, Z., Kondo, S., Kanegae, Y. & Saito, I. Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: Application to vector titration. Biochem. Biophys. Res. Commun. 417, 945\u2013950. https:\/\/doi.org\/10.1016\/j.bbrc.2011.12.016 (2012).","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"3803_CR60","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot095117","author":"MR Green","year":"2018","unstructured":"Green, M. R. & Sambrook, J. The basic polymerase chain reaction (PCR). Cold Spring. Harb. Protoc. https:\/\/doi.org\/10.1101\/pdb.prot095117 (2018).","journal-title":"Cold Spring. Harb. Protoc."},{"key":"3803_CR61","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1007\/978-1-60761-753-2_15","volume":"649","author":"DY Guschin","year":"2010","unstructured":"Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247\u2013256. https:\/\/doi.org\/10.1007\/978-1-60761-753-2_15 (2010).","journal-title":"Methods Mol. Biol."},{"key":"3803_CR62","doi-asserted-by":"publisher","first-page":"559","DOI":"10.1111\/j.1348-0421.2005.tb03753.x","volume":"49","author":"Y Baba","year":"2005","unstructured":"Baba, Y., Nakano, M., Yamada, Y., Saito, I. & Kanegae, Y. Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells. Microbiol. Immunol. 49, 559\u2013570. https:\/\/doi.org\/10.1111\/j.1348-0421.2005.tb03753.x (2005).","journal-title":"Microbiol. Immunol."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-03803-w.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-03803-w","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-03803-w.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,3]],"date-time":"2022-12-03T18:33:47Z","timestamp":1670092427000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-03803-w"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,21]]},"references-count":62,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["3803"],"URL":"https:\/\/doi.org\/10.1038\/s41598-021-03803-w","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,21]]},"assertion":[{"value":"13 September 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"10 December 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"21 December 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"24369"}}