biostudies-literatureUnknown31Kostyushev DCRISPR-Cas9 systems can directly target the hepatitis B virus (HBV) major genomic form, covalently closed circular DNA (cccDNA), for decay and demonstrate remarkable anti-HBV activity. Here, we demonstrate that CRISPR-Cas9-mediated inactivation of HBV cccDNA, frequently regarded as the "holy grail" of viral persistence, is not sufficient for curing infection. Instead, HBV replication rapidly rebounds because of <i>de novo</i> formation of HBV cccDNA from its precursor, HBV relaxed circular DNA (rcDNA). However, depleting HBV rcDNA before CRISPR-Cas9 ribonucleoprotein (RNP) delivery prevents viral rebound and promotes resolution of HBV infection. These findings provide the groundwork for developing approaches for a virological cure of HBV infection by a single dose of short-lived CRISPR-Cas9 RNPs. Blocking cccDNA replenishment and re-establishment from rcDNA conversion is critical for completely clearing the virus from infected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.Molecular therapy. Nucleic acids482-493https://www.ebi.ac.uk/biostudies/studies/S-EPMC9972396biostudies-literatureDepleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9.PMC9972396Kostyusheva AEgorshina ASudina ADunaeva EZakirova NFBayurova EKostyushev DGoptar IYanvarev DVPonomareva NNikiforova ALukashev AGordeychuk IBrezgin SAbramov IFrolova ALisitsa TZamyatnin AAChulanov VIvanov AfalseDepleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9.CRISPR-Cas9 systems can directly target the hepatitis B virus (HBV) major genomic form, covalently closed circular DNA (cccDNA), for decay and demonstrate remarkable anti-HBV activity. Here, we demonstrate that CRISPR-Cas9-mediated inactivation of HBV cccDNA, frequently regarded as the "holy grail" of viral persistence, is not sufficient for curing infection. Instead, HBV replication rapidly rebounds because of <i>de novo</i> formation of HBV cccDNA from its precursor, HBV relaxed circular DNA (rcDNA). However, depleting HBV rcDNA before CRISPR-Cas9 ribonucleoprotein (RNP) delivery prevents viral rebound and promotes resolution of HBV infection. These findings provide the groundwork for developing approaches for a virological cure of HBV infection by a single dose of short-lived CRISPR-Cas9 RNPs. Blocking cccDNA replenishment and re-establishment from rcDNA conversion is critical for completely clearing the virus from infected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.2023-01-01T00:00:00Z2023 Mar2024-11-19T16:01:54.764Z2024-11-19T16:01:54.764ZS-EPMC99723963686508910.1016/j.omtn.2023.02.001