Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System

Abstract

The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.

Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg‐positive patients with functional cure following PEG‐IFN‐based therapy

“…This is consistent with the architecture of the integrated HBV DNA observed by long‐read sequencing, which comprises the entire HBV genome, beginning and ending with the DR1 26 . Long‐read sequencing has better performance in showing the flanking region and multiple HBV splice variants than short‐read sequencing, 26,36 which should be considered in future research. The splice donor site (GT) at nt 458 formed a second breakpoint peak in the chimeric transcript analysis, which supported a previous study showing that the host splicing machinery may use foreign splice sites of the HBV genome during post‐transcriptional pre‐mRNA splicing 14 …”

“…34,35 This is consistent with the architecture of the integrated HBV DNA observed by long-read sequencing, which comprises the entire HBV genome, beginning and ending with the DR1. 26 Long-read sequencing has better performance in showing the flanking region and multiple HBV splice variants than short-read sequencing, 26,36 which should be considered in future research.…”

A versatile method to profile hepatitis B virus DNA integration

“…RAISING-sequencing of the HBV-HCC-derived cell lines, PLC/PRF/5 and Hep3B cells, revealed viral integration profiles consistent with previous reports. 28 – 30 In addition, the assay could determine de novo HBV DNA integration in the early stages of infection in HepG2-hNTCP-C4 cells and primary human hepatocytes, in line with recent studies. 32 – 34 Furthermore, RAISING-sequencing successfully identified the chimeric sequences in HBV-infected tissues, including humanized livers and clinical specimens.…”

The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19

“…Chronic infection with HBV with a persistently elevated level of HBV surface antigen (HBsAg) is considered another critical risk factor for the development of HCC. Interestingly, the CRISPR/Cas9 strategy revealed a powerful effect in destroying the intrahepatic HBV genome and reducing HBsAg levels in vitro and in animal models ( Lin et al., 2014 ; Chen et al., 2021 ). In one study, CRISPR/Cas9 was designed with specific single-guide RNAs (sgRNAs) to knock out the expression of HBsAg by targeting the open reading frames, preS1/preS2/S, of the HBV gene in the HCC cell line.…”