Z Gastroenterol 2015; 53 - A5_9
DOI: 10.1055/s-0035-1568102

CRISPR/Cas9 “double”-nickase mediated inactivation of hepatitis B virus replication

M Karimova 1, N Beschorner 1, W Dammermann 2, J Chemnitz 1, D Indenbirken 1, A Grundhoff 1, S Lüth 2, F Buchholz 4, J Schulze zur Wiesch 2, J Hauber 1
  • 1Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Hamburg, Germany
  • 2University Medical Center Eppendorf, 1. Department of Medicine, Hamburg, Germany
  • 3German Center for Infection Research (DZIF), Partner site Hamburg, Hamburg, Germany
  • 4University Hospital and Medical Faculty Carl Gustav Carus, Department of Medical Systems Biology, Dresden, Germany

Background:

Current antiviral therapies cannot cure hepatitis B virus (HBV) infection, since successful HBV eradication would require the inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer-enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, provide the technology for the development of advanced therapy strategies that directly at-tack the HBV genome.

Methods:

We report here the identification of cross-genotype conserved HBV sequences in the HbS and HbX region of the HBV genome that are specifically and effectively inactivated by a Cas9 double-nickase approach. Pairs of appropriately spaced Cas9 nickase mutants introduced two single-strand breaks on the opposite DNA strands that were subject to NHEJ repair in order to avoid off-target mutations by improving specificity by up to 1,500-fold relative to the wild-type Cas9 enzyme.

Results:

We show that this approach equally inactivated episomal cccDNA as well as chromosomally integrated HBV target sites in reporter cell lines as well as in chronically infected hepatoma cell lines. Analysis of Cas9n activity on ORF S and X target sites by next generation sequencing revealed efficient editing of cccDNA molecules targeted by either gRNA in HBV-infected HepG2.2.15, HepG2-H1.3 and HepG2-NTCP cells. The efficiency of X-specific sgRNAs was particularly high, with approximately 90% of all amplicons reads showing clearly discernible indel signatures.

Conclusion:

Our data support the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to provide a cure for HBV infection.

Corresponding author: Dammermann, Werner

E-Mail: w.dammermann@uke.de