Depleting 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 de novo 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 in-fected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.

Automated summary

CRISPR-Cas9 systems show remarkable anti-HBV activity by targeting the hepatitis B virus major genomic form, cccDNA. However, inactivating cccDNA alone is not enough to cure the infection, as HBV replication rebounds due to the de novo formation of cccDNA from its precursor, rcDNA. Depleting rcDNA before delivering CRISPR-Cas9 RNPs can prevent viral rebound and promote resolution of HBV infection. These findings lay the groundwork for developing a virological cure of HBV infection using a single dose of short-lived CRISPR-Cas9 RNPs, with the help of widely used reverse transcriptase inhibitors to block cccDNA replenishment.