Introduction
It originates from the adaptive
Over 250 million people worldwide are chronically
infected by the hepatitis B virus (HBV). Symptoms
of HBV are fatigue, fever, nausea, joint pain,
abdominal pain and yellowing of the skin and eyes.
Prolonged HBV infection can lead to severe liver
failure, cirrhosis, and hepatocellular liver cancer. It
can be fatal when not treated. Up to 600.000
people a year are likely to die from a hepatitis B
infection (Dienstag et al., 2008).
HBV is a double-stranded DNA virus of the
Hepadnaviridae family. The virus binds to the
hepatocyte membrane in the liver after which the
HBV core is being released in the cytoplasm and
subsequently transported to the nucleus where it
will form a covalently closed circular DNA (cccDNA)
(figure 1). This cccDNA is crucial for the
persistence of the infection, since it serves as a
template for transcription of all viral genomes and
sub-genomes.
Figure 1. Schematic overview of the HBV life cycle.
Treatment of hepatitis B is currently based on
inhibiting viral replication by acting on post-
transcriptionally processes (Schlomai & Rice,
2014). Consequently, cccDNA is not targetted, so it
remains stable and persists in the hepatocyte’s
nucleus. Hence, medication must be taken
chronically to prevent further viral outbreaks.
Infected liver cells may contain up to 50 cccDNA
molecules (Balsano et al., 2008), therefore agents
acting directly on cccDNA might be more desirable
and possibly even curative. For example, specific
nucleases that are able to break the viruses
double-stranded DNA, without damaging the host
genomic DNA, would be ideal for this purpose.
The recently developed CRISPR-Cas9
technology makes it possible to manipulate DNA
very specifically.
immune system of bacteria against viruses and
plasmids. When a virus enters a bacterium, it will
insert its own DNA in the host DNA for further
replication. Some bacteria are able to copy a short
sequence of the virus’s DNA (target sequence), to
serve as a detection mechanism for future viral
infections. The complex in which the detection
mechanism operates is called CRISPR. The CRISPR
complex is attached to the nuclease Cas9. In short,
CRISPR is able to recognize the viral DNA through
its target sequence and Cas9, in turn, is able to
destroy the viral DNA by breaking the double helix.
Genome engineering has made it possible to
design target sequences of CRISPR, which is called
guide RNA (gRNA). So, in principle any desired
DNA sequence can be recognized and cleaved by
CRISPR-Cas9. Moreover, extra nucleotides can be
inserted to CRISPR-Cas9 and accordingly built in
into the cell’s genome. Therefore CRISPR-Cas9 can
be applied to transform DNA that causes life
threatening diseases.
In this study we want to investigate the
application of CRISPR-Cas9 in the treatment of
hepatitis B by direct targeting and cleavage of HBV
cccDNA. We hypothesize that HBV can be
suppressed by CRISPR-Cas9 through cleavage of
the HBV genome. Hence, we first examine the
efficiency of several gRNAs in HBV infected cell
lines by measuring an important indicator for viral
expression and replication in the medium,
specifically the secretion of surface antigen HBsAg.
Next, we sought to evaluate the antiviral effect of
the most effective gRNAs in a mouse model of
HBV, to ensure that it functions properly in the
hepatocytes of a living organism. Finally, we
wanted to examine the long-term anti-HBV effect
of CRISPR-Cas9 in a special cell line that mimics
the HBV life cycle components and consequently
produces the infectious virus (Sells et al., 1987).
Therefore, we measured the amount of cccDNA at
day 24 and 34 after treatment. In all experiments
we expect CRISPR-Cas9 to cause a decrease in
HBV expression compared to control conditions.
Materials and Methods
CRISPR-Cas9 design and validation
Whole-genome sequences from HBV were inquired
from the GenBank sequence database to
determine several target sequences. We made use
of a CRISPR online design tool Dharmacon
(https://horizondiscovery.com/en/resources/featur Hydrodynamic injection of
ed-articles/dharmacon-editr-crispr-cas9-gene- 1.3x WT HBV and Cas9/gRNA
engineering-system) to generate and order four
different gRNAs (table 1), that were able to target
the HBV DNA at different locations. To evaluate the
efficiency of the gRNA’s we added the four
different designs of CRISPR-Cas9 to HBV infected
cell lines, that were maintained in DMEM and 10%
calf serum. We used mismatching target
sequences as control. To determine the efficiency
Draw blood to analyze
of the gRNA after 72 hours, we measured an
serum virus load
indicator of HBV genomic expression in the
Figure 2. Schematic overview of in vivo experiment.
medium of the cell lines, namely hepatitis B
surface antigen (HBsAg). The two most efficient
Statistical analyses
gRNAs were used for follow-up experiments.
All analyses were performed in SPSS 14.0.
Table 1. Sequences of the manufactured gRNAs. Comparisons between two groups were analyzed
by Students t-test. Comparisons among more
groups were analyzed by one-way ANOVA followed
by Tukey’s post-hoc test to compare between two
groups.

Anti-HBV effect in vivo

We used a mouse model of HBV to evaluate the
anti-viral effect of CRISPR-Cas9 in vivo. NRG mice
were administered 15 µg 1,3xHBV plasmid and 20
µg CRISPR-Cas9 through tail vain hydrodynamic
injection in 7-9 seconds (figure 2). We took
measures on HBV titer in a blood sample to
determine the anti-viral effects of CRISPR-Cas9 at
day 2 and 4 post injection. Mismatching target
sequences were used as control. Mice were
obtained from Jackson laboratory and were housed
in an accredited facility with ad libitum access to
food and water. All animal protocols were
approved by the University of Massachusetts.

Long term anti-HBV effect

For this experiment we used the HepG2.2.15
hepatoblastoma cell line, which contains HBV
cccDNA and is able to produce infectious virions. In
contrast to other cell lines, HepG2.2.15 is ideal for
taking measures of cccDNA. We measured the
effect of CRISPR-Cas9 mediated cleavage on the
abundance of cccDNA in cells after 24 and 34 days.
Control constructs were made of mismatching
target sequences.