Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA

Abstract

Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.

Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

“…Seeger et al demonstrated that the use of CRISPR/Cas9 is currently the best method of functionally inactivating HBV cccDNA (Seeger and Sohn, 2016). HBV infection can induce HBV DNA fragment integration into the host genome, which had been observed in ~80% of cases of HBV-induced hepatocarcinogenesis (Bréchot, 2004; Hai et al, 2014).…”

Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B

“…A series of studies have confirmed the therapeutic effectiveness of this editing tool in targeting HBV DNA both in vitro and in vivo (Kennedy et al, 2015; Lin et al, 2014; Moyo et al, 2017; Ramanan et al, 2015; Seeger and Sohn, 2014, 2016). However, therapeutic use of CRISPR/Cas9 faces significant challenges with respect to delivery, efficiency and safety.…”

The current status and future directions of hepatitis B antiviral drug discovery

“…Therefore, several sequence-specific DNA editing technologies, such as zinc finger nucleases, meganucleases, transcription activator-like effector nuclease (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 57–62 , have been explored as potential therapeutic approaches to functionally inactivate or eliminate cccDNA. Recently, using next generation sequencing (NGS) technology, Seeger and Sohn determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ) DNA repair pathway 63 . The authors elegantly demonstrated that over 90% of HBV cccDNA was edited by Cas9.…”

“…Moreover, approximately 6% of the cleavage cccDNA had deletions of 3 and 6 nucleotides which maintain the open reading frame of HBx protein. If the one or two amino acid deletion does not compromise HBx function, the results suggest that in order to completely inactivate cccDNA function, more than one sgRNAs targeting cccDNA sequences encoding critical residues of HBV proteins are desired 63 . Moreover, double cleavage of cccDNA by Cas9 may not only more efficiently inactivate cccDNA function, but also make cccDNA repair more difficult and thus favor its elimination 64 .…”