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.
Highlights
Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers which currently ranks the fourth leading cause of cancer-related deaths worldwide (Petrick et al, 2020)
The DNA extracted from PLC/PRF/5 is sequenced by the Pacbio platform, and the correction module of the Canu software was used for row reads correction
Using CRISPR/ Cas9 system, we knocked out the integrated hepatitis B virus (HBV) S fragment and decreased HBsAg level in PLC/PRF/5 cells, suggested the potential use of CRISPR/Cas9 genome editing technique to cut integrated HBV DNA, as well as for function cure of chronic hepatitis B patients
Summary
Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers which currently ranks the fourth leading cause of cancer-related deaths worldwide (Petrick et al, 2020). The integration of HBV in the human genome is considered to be one of the key mechanisms of HBV carcinogenesis (D’Souza et al, 2020). A series of studies confirmed HBV DNA is randomly inserted into the human genome in HBV-infected liver tissues, including acute hepatitis B, chronic hepatitis B, cirrhosis and especially HCC (Jiang et al, 2012; Li et al, 2014). With the development of next-generation sequencing (NGS), many studies have achieved genome-wide surveys of HBV integration in HCC, and a great deal of data on the HBV pathogen in HCC has been obtained. The third-generation sequencing (TGS) technology developed in recent years has extremely high sequencing throughput, and generate reads up to 60 kbp long (Rhoads and Au, 2015), showing a good potential for detecting HBV integration
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