Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Abstract

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters

“…Indicator plasmids consisting of these viral DNA target sequences inserted 3 ′ to a translation initiation codon and 5 ′ to the firefly luciferase (FLuc) gene, constructed as previously described (Kennedy et al , 2015, were then cotransfected into 293T cells along with a plasmid encoding Spy Cas9 and an sgRNA linked to either a tRNA or U6 promoter as well as a Renilla luciferase (RLuc)-based internal control plasmid. Cells were harvested at 72 h post-transfection and FLuc and RLuc levels determined.…”

Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome

“…Due to the high heterogeneity of the HBV genome, HBV was defined as different genotypes or subgenotypes with a sequence divergence w8 % or w4 %, respectively. During the preparation of our manuscript, several papers related to harnessing HBV with CRISPR/Cas9 systems were published (Kennedy et al, 2014;Lin et al, 2014;Seeger & Sohn, 2014;Zhen et al, 2015). However, the high heterogeneity of the HBV genome was not considered during the selection of their gRNA targets.…”

“…ZFNs, TALENs and CRISPR/Cas9 systems. Although targeting the HBV genome by these genome-editing technologies has been reported previously (Bloom et al, 2013;Kennedy et al, 2014;Lin et al, 2014;Seeger & Sohn, 2014;Weber et al, 2014;Zhen et al, 2015), here we would like to propose a way to inhibit HBV with different genotypes by using the same gRNA/Cas9 system, and explore the possibility and efficiency of multiple gRNAs/ Cas9 systems. With this study, we provide the possibility to inhibit viral replication and clear the cccDNA of HBV of different genotypes, which might be a functional anti-HBV therapy to avoid the mutations during viral replication.…”

Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression

“…5,21) In addition, a CRISPR/Cas9 system targeting the hepatitis B virus (HBV) genome efficiently inhibited the viral replication. 22,23) In particular, the CRISPR/Cas9 system is promising for cleavage of HBV covalently closed circular DNA (cccDNA), which plays a crucial role in persistent HBV infection and is an important target for anti-HBV drugs; however, there are no drugs which can eliminate cccDNA from the cells. In the future, we should pay close attention to the application of CRISPR/Cas9 systems to HBV treatment.…”