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The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)\/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)\/Cas9\u2010ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR\/Cas9\u2010mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV\u2010infected human hepatoma cell line HepG2.2.15 with HBV\u2010specific gRNA\/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and\/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene\u2010editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off\u2010target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA\/Cas9 RNP\u2010based gene\u2010editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.<\/jats:p>","DOI":"10.1002\/jmv.28952","type":"journal-article","created":{"date-parts":[[2023,7,17]],"date-time":"2023-07-17T02:39:00Z","timestamp":1689561540000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Synthetic gRNA\/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication"],"prefix":"10.1002","volume":"95","author":[{"given":"Jinyu","family":"Zhang","sequence":"first","affiliation":[{"name":"Center of Excellence in Inflammation, Infectious Disease and Immunity, Quillen College of Medicine East Tennessee State University Johnson City Tennessee USA"},{"name":"Department 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Tennessee State University Johnson City Tennessee USA"},{"name":"Department of Internal Medicine Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU Johnson City Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed","family":"El Gazzar","sequence":"additional","affiliation":[{"name":"Center of Excellence in Inflammation, Infectious Disease and Immunity, Quillen College of Medicine East Tennessee State University Johnson City Tennessee USA"},{"name":"Department of Internal Medicine Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU Johnson City Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan P.","family":"Moorman","sequence":"additional","affiliation":[{"name":"Center of Excellence in Inflammation, Infectious Disease and Immunity, Quillen College of Medicine East Tennessee State University Johnson City Tennessee 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