CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

The CRISPR-Cas system has first been discovered in bacteria and archaea as a defence
mechanism for invading viruses. This system can specifically target and cleave conserved region of
the invading virus resulting to suppression of viral gene expression and replication. The type II
CRISPR-Cas system of Streptococcus pyogenes SF 370 has been adapted as an RNA-guided sequence
specific DNA nuclease for use in mammalian cells to completely suppress or degrade viruses such as
the hepatitis B virus (HBV). The HBV genome exists in the nuclei of infected hepatocytes as a 3.2 kb
double-stranded episomal DNA. The persistence of HBV in infected hepatocytes is due to the
presence of covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs.
Antiviral therapies can inhibit replication of HBV DNA in capsids present in the cytoplasm, but do not
reduce or destroy nuclear cccDNA of infected cells. Hence, these developed antiviral therapies are
effective only in suppressing but not eliminating HBV infections. The current study aims to explore
the application of CRISPR/Cas9 system to target HBV DNA in mammalian cells hoping that by directly
targeting the HBV genome could suppress HBV or decreasing the stability of cccDNa.

The study was conducted by co-transfecting the HepG hepatoma cell line with a HBV-
expressing plasmid and constructs expressing Cas9 and with the chosen single guide RNAs (sgs 14,
17, 19 and 21). Indicators for viral gene expression and replication were then measured to evaluate
the efficacy of the selected sgRNAs in targeting HBV. The antiviral effect of Cas9 on primary
hepatocytes was evaluated by introducing the HBV and Cas9/sg21 plasmids to the liver of
immunodeficient mice (NRG) by hydrodynamic injection (HDI). The efficacy of sustained Cas9/sgRNA
expression in inhibiting HBV was evaluated in HEPG2.2.15 hepatoblastoma cell line and HepG2 cells
overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (Hep-NTCP) was
used to evaluate the anti-HBV CRISPR/Cas9 in de novo infection.

Among the chosen single guide RNAs, sg 17 and sg21 resulted in a decrease in HBV 3.5kb
RNA levels and secretion of surface antigen (HBsAg). In addition, the combination of two RNA guides
(sg17 and sg 21) in targeting HBV is more effective in reducing both indicators for viral gene
expression and replication (HBV 3.5kb RNA and HBsAg) as compared to the single guide RNAs. The
antiviral effect of Cas9/sg21 showed suppression of HBV expression as compared to NRG controls
expressing Cas9 and a mutated sgRNA (sg21M; 3’5 bp mismatch). Transduced HepG2.2.15 cells with
Cas9-2A Puro lentiviruses encoding Cas9 and individual sgRNAs (sg6, sg17, sg21) showed dramatic
suppression of HBV DNA release, HBeAg secretion and production of mRNA as compared to control.
Experimentation to evaluate the anti-HBV CRISPR/Cas9 in inhibiting de novo HBV infection provides
direct evidence that Cas9 is capable of targeting episomal forms of the virus, and exerting anti-HBV
effects by directly targeting cccDNA.

Taken together, this study was able to show that targeting multiple conserved region of HBV
with Cas9 results in robust suppression of viral replication and direct mutagenesis and depletion of
cccDNA. The unique advantages of the CRISPR/Cas9 system (such as multiplex targeting) are of
interest in developing antiviral applications. Our work provides an extension beyond these
complementary studies, by demonstrating the anti-HBV effect of sgRNAs specifically targeting highly
conserved regions of HBV in vitro and in vivo, by directly confirming mutagenesis in cccDNA in a de
novo infection model of HBV, and extending this antiviral activity to patient-derived virus.
Additionally our finding that appropriately chosen virus-targeting sgRNAs can avoid inducing
off-target cleavage, even upon sustained Cas9/sgRNA expression, strengthens the case for selecting
viral targets as good candidates for CRISPR/Cas9 therapeutic use.