Adapting the staphylococcus aureus CRISPR/Cas9 gene editing system to target hepatitis B virus

Abstract

Chronic hepatitis B virus (HBV) infection remains a global health burden despite the availability of approved therapeutics and a vaccine. Complications associated with chronic HBV infection include cirrhosis and hepatocellular carcinoma. Current approved therapies are not completely effective for the treatment of chronic HBV infection, as these do not target the covalently closed circular DNA (cccDNA) of the virus. The cccDNA intermediate is required for the transcription of viral RNA resulting in the persistence of the infection. Several designer nucleases have been explored as a promising option to target the cccDNA to achieve a sustainable therapy. This is based on the ability to introduce double stranded breaks (DSBs) at a specific DNA target site. A DSB may lead to the non-homologous end joining (NHEJ) repair pathway which is error-prone and introduces mutations in the target gene. Based on this rationale, this project focused on the adaptation of the Staphylococcus aureus CRISPR/Cas9 system to target various regions of the HBV genome with the aim of disrupting the cccDNA. This RNA-guided nuclease system requires the Cas9 nuclease, a single guide RNA (sgRNA) sequence as well as the protospacer adjacent motif (PAM) sequence for target recognition. To achieve targeted disruption, a portion of the sgRNA sequence called the CRISPR RNA (crRNA) was designed using in silico tools to target all the open reading frames of the HBV genome. Immunofluorescence staining and RT-qPCR in vitro analysis confirmed the successful expression of the Cas9 nuclease and crRNA sequences. However, in vitro analysis confirmed the newly designed crRNA sequences did not achieve significant targeted disruption of the cccDNA. In vitro studies also confirmed the SaCas9 nuclease did not exhibit any knockdown effect on the HBV replication marker, hepatitis B surface antigen (HBsAg). Although the crRNA sequences were not efficient, these sequences were not associated with any cytotoxicity to the cells. It was therefore concluded that the CRISPR/Cas9 system depends largely on the design of the crRNA sequences as these are crucial for the Cas9 targeted cleavage abilities. Further improvements in the design of these artificial nuclease systems will be carried out to improve their efficiency to treat HBV.