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

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

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1652-1663
Number of pages12
JournalHepatology Communications
Volume6
Issue number7
DOIs
StatePublished - Jul 2022

Bibliographical note

Funding Information:
American Cancer Society (ISG-16-210-01-RMC)

Publisher Copyright:
© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.

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