CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X.
Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require 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 technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off-target effects. Here, we identified cross-genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection.
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.
Madina
M
Karimova
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.
Niklas
N
Beschorner
Department of Medicine I, University Medical Center Eppendorf, 20246 Hamburg, Germany.
Werner
W
Dammermann
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.
Jan
J
Chemnitz
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.
Daniela
D
Indenbirken
Department of Medicine I, University Medical Center Eppendorf, 20246 Hamburg, Germany.
Jan-Hendrik
JH
Bockmann
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.|German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany.
Adam
A
Grundhoff
Department of Medicine I, University Medical Center Eppendorf, 20246 Hamburg, Germany.
Stefan
S
Lüth
Department of Medical Systems Biology, University Hospital and Medical Faculty Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany.
Frank
F
Buchholz
Department of Medicine I, University Medical Center Eppendorf, 20246 Hamburg, Germany.|German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany.
Julian
J
Schulze zur Wiesch
Heinrich Pette Institute - Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany.|German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany.
Joachim
J
Hauber
D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000235:genetics*; D020868:Gene Silencing; D016679:Genome, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*
D003850:Deoxyribonuclease I