{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,4]],"date-time":"2026-02-04T17:58:35Z","timestamp":1770227915099,"version":"3.49.0"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,6,26]],"date-time":"2021-06-26T00:00:00Z","timestamp":1624665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR\/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR\/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR\/<jats:italic>Staphylococcus aureus<\/jats:italic> Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR\/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic>. Three functional guide RNAs (gRNAs), T<jats:sub>2<\/jats:sub>, T<jats:sub>3<\/jats:sub>, and T<jats:sub>6<\/jats:sub>, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR\/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.<\/jats:p>","DOI":"10.3389\/fmicb.2021.665184","type":"journal-article","created":{"date-parts":[[2021,6,26]],"date-time":"2021-06-26T04:11:25Z","timestamp":1624680685000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":25,"title":["Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR\/SaCas9 Expressed From Liver-Specific Promoters"],"prefix":"10.3389","volume":"12","author":[{"given":"Kun","family":"Yan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiangpeng","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongyun","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiaohong","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiali","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tianmo","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Muhammad","family":"Sajid","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hafiz","family":"Ullah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Limin","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,6,26]]},"reference":[{"key":"ref1","first-page":"1028a","article-title":"Analysis of the effect on HBV life cycle by HBV genome editing using TALEN and CRISPR\/Cas9 systems","volume":"60","author":"Abe","year":"2014","journal-title":"Hepatology"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"416","DOI":"10.1038\/s41586-018-0500-9","article-title":"In vivo CRISPR editing with no detectable genome-wide off-target mutations","volume":"561","author":"Akcakaya","year":"2018","journal-title":"Nature"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"86","DOI":"10.2174\/1566523214666140302193709","article-title":"Basic biology of adeno-associated virus (AAV) vectors used in gene therapy","volume":"14","author":"Balakrishnan","year":"2014","journal-title":"Curr. Gene Ther."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1111\/j.1440-1746.2010.06514.x","article-title":"Significance of hepatitis B virus genotypes and mutations in the development of hepatocellular carcinoma in Asia","volume":"26","author":"Chan","year":"2011","journal-title":"J. Gastroenterol. Hepatol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"40","DOI":"10.3390\/v10010040","article-title":"CRISPR-Cas targeting of host genes as an antiviral strategy","volume":"10","author":"Chen","year":"2018","journal-title":"Viruses"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1017","DOI":"10.1111\/j.1872-034X.2011.00873.x","article-title":"Hepatitis B virus drug resistance to current nucleos(t)ide analogs: mechanisms and mutation sites","volume":"41","author":"Deng","year":"2011","journal-title":"Hepatol. Res."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1016\/j.antiviral.2015.03.015","article-title":"Targeting hepatitis B virus cccDNA by CRISPR\/Cas9 nuclease efficiently inhibits viral replication","volume":"118","author":"Dong","year":"2015","journal-title":"Antivir. Res."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"767","DOI":"10.1016\/0092-8674(86)90519-2","article-title":"Tissue-specific in vitro transcription from the mouse albumin promoter","volume":"47","author":"Gorski","year":"1986","journal-title":"Cell"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"25552","DOI":"10.1038\/srep25552","article-title":"The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely","volume":"6","author":"Guo","year":"2016","journal-title":"Sci. Rep."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"510","DOI":"10.1006\/jsre.1994.1082","article-title":"Liver-directed gene therapy: evaluation of liver specific promoter elements","volume":"56","author":"Hafenrichter","year":"","journal-title":"J. Surg. Res."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"3394","DOI":"10.1182\/blood.V84.10.3394.bloodjournal84103394","article-title":"Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes","volume":"84","author":"Hafenrichter","year":"","journal-title":"Blood"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"4345","DOI":"10.1128\/JVI.03094-14","article-title":"Inhibition of hepatitis B virus gene expression and replication by hepatocyte nuclear factor 6","volume":"89","author":"Hao","year":"2015","journal-title":"J. Virol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1007\/978-1-4939-9145-7_13","article-title":"Use of CRISPR\/Cas9 for the modification of the mouse genome","volume":"1953","author":"Klimke","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"445","DOI":"10.1038\/nrg3742","article-title":"Engineering adeno-associated viruses for clinical gene therapy","volume":"15","author":"Kotterman","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"375","DOI":"10.1016\/S1525-0016(02)00060-6","article-title":"In vitro and in vivo comparative study of chimeric liver-specific promoters","volume":"7","author":"Kramer","year":"2003","journal-title":"Mol. Ther."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"36483","DOI":"10.1038\/srep36483","article-title":"Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs","volume":"6","author":"Li","year":"2016","journal-title":"Sci. Rep."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"2080","DOI":"10.3389\/fimmu.2018.02080","article-title":"Inhibition of HBV expression in HBV transgenic mice using AAV-delivered CRISPR-SaCas9","volume":"9","author":"Li","year":"","journal-title":"Front. Immunol."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"56","DOI":"10.1002\/hep.29406","article-title":"Recombinant covalently closed circular DNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model","volume":"67","author":"Li","year":"","journal-title":"Hepatology"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"e186","DOI":"10.1038\/mtna.2014.38","article-title":"The CRISPR\/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo","volume":"3","author":"Lin","year":"2014","journal-title":"Mol. Ther. Nucl. Acids"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"2252","DOI":"10.1099\/vir.0.000159","article-title":"Inhibition of hepatitis B virus by the CRISPR\/Cas9 system via targeting the conserved regions of the viral genome","volume":"96","author":"Liu","year":"2015","journal-title":"J. Gen. Virol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1016\/j.antiviral.2018.02.011","article-title":"Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus","volume":"152","author":"Liu","year":"2018","journal-title":"Antivir. Res."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"370","DOI":"10.1016\/j.jhep.2017.03.021","article-title":"EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection","volume":"67","author":"Liver","year":"2017","journal-title":"J. Hepatol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1902312","DOI":"10.1002\/advs.201902312","article-title":"CRISPR\/Cas systems in genome editing: methodologies and tools for sgRNA design, off-target evaluation, and strategies to mitigate off-target effects","volume":"7","author":"Manghwar","year":"2020","journal-title":"Adv. Sci."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"22371","DOI":"10.1016\/S0021-9258(18)45715-8","article-title":"Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)\/bovine growth hormone gene in transgenic mice","volume":"265","author":"McGrane","year":"1990","journal-title":"J. Biol. Chem."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1126\/science.abb1400","article-title":"CRISPR-Cas phi from huge phages is a hypercompact genome editor","volume":"369","author":"Pausch","year":"2020","journal-title":"Science"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1281","DOI":"10.1016\/j.jhep.2017.07.011","article-title":"How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach?","volume":"67","author":"Rajoriya","year":"2017","journal-title":"J. Hepatol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"1766","DOI":"10.1128\/MCB.3.10.1766","article-title":"Transcription of hepatitis-B virus by Rna polymerase-Ii","volume":"3","author":"Rall","year":"1983","journal-title":"Mol. Cell. Biol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1038\/nature14299","article-title":"In vivo genome editing using Staphylococcus aureus Cas9","volume":"520","author":"Ran","year":"2015","journal-title":"Nature"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1016\/S2468-1253(19)30119-0","article-title":"A global scientific strategy to cure hepatitis B","volume":"4","author":"Revill","year":"2019","journal-title":"Lancet Gastroenterol. Hepatol."},{"key":"ref30","first-page":"1002","article-title":"HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene","volume":"3","author":"Sandig","year":"1996","journal-title":"Gene Ther."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"7401","DOI":"10.1038\/s41598-017-07642-6","article-title":"ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells","volume":"7","author":"Scott","year":"2017","journal-title":"Sci. Rep."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"m2200","DOI":"10.1136\/bmj.m2200","article-title":"Hepatitis B virus persistence and reactivation","volume":"370","author":"Shi","year":"2020","journal-title":"BMJ"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.1016\/S0140-6736(16)30579-7","article-title":"The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013","volume":"388","author":"Stanaway","year":"2016","journal-title":"Lancet"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1002\/hep.28156","article-title":"AASLD guidelines for treatment of chronic hepatitis B","volume":"63","author":"Terrault","year":"2016","journal-title":"Hepatology"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"440","DOI":"10.1096\/fasebj.8.6.8168695","article-title":"Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations","volume":"8","author":"Valera","year":"1994","journal-title":"FASEB J."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1186\/s12977-017-0375-0","article-title":"Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection","volume":"14","author":"Wang","year":"2017","journal-title":"Retrovirology"},{"key":"ref37","doi-asserted-by":"publisher","first-page":"159","DOI":"10.21037\/atm.2017.03.69","article-title":"Alpha-interferon treatment in hepatitis B","volume":"5","author":"Woo","year":"2017","journal-title":"Ann. Transl. Med."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1149","DOI":"10.1016\/j.jhep.2017.02.004","article-title":"HBVcircle: a novel tool to investigate hepatitis B virus covalently closed circular DNA","volume":"66","author":"Yan","year":"2017","journal-title":"J. Hepatol."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1007\/978-1-4939-9139-6_7","article-title":"Use of AAV vectors for CRISPR-mediated in vivo genome editing in the retina","volume":"1950","author":"Yu","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1016\/j.cell.2015.09.038","article-title":"Cpf1 is a single-RNA-guided endonuclease of a Class 2 CRISPR-Cas system","volume":"25","author":"Zetsche","year":"2016","journal-title":"Transgenic Res."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"884","DOI":"10.1111\/pbi.13259","article-title":"Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system","volume":"18","author":"Zhang","year":"2020","journal-title":"Plant Biotechnol. J."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"162","DOI":"10.1016\/j.virusres.2010.01.012","article-title":"Evaluation of hepatitis B virus promoters for sustained transgene expression in mice by bioluminescence imaging","volume":"149","author":"Zhao","year":"2010","journal-title":"Virus Res."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1016\/j.coviro.2016.06.004","article-title":"Current treatments for chronic hepatitis B virus infections","volume":"18","author":"Zoulim","year":"2016","journal-title":"Curr. Opin. Virol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"1593","DOI":"10.1053\/j.gastro.2009.08.063","article-title":"Hepatitis B virus resistance to nucleos(t)ide analogues","volume":"137","author":"Zoulim","year":"2009","journal-title":"Gastroenterology"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.665184\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,6,26]],"date-time":"2021-06-26T04:11:30Z","timestamp":1624680690000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.665184\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,26]]},"references-count":44,"alternative-id":["10.3389\/fmicb.2021.665184"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2021.665184","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,26]]},"article-number":"665184"}}