Hepatology Communications (Jul 2022)

Efficient silencing of hepatitis B virus S gene through CRISPR‐mediated base editing

  • Hao Zhou,
  • Xiaomei Wang,
  • Clifford J. Steer,
  • Guisheng Song,
  • Junqi Niu

DOI
https://doi.org/10.1002/hep4.1933
Journal volume & issue
Vol. 6, no. 7
pp. 1652 – 1663

Abstract

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Abstract Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non‐homologous end‐joining repair of double‐stranded break (DSB). However, the CRISPR/Cas9‐mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single‐guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5‐CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5‐CBE cells. Phenotypically, 71% of PLC/PRF/5‐CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5‐CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off‐target effect was detected in predicted off‐target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR‐mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.