Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Chia-Chen, Guan, Guiwen, Qi, Xuewei, Abulaiti, Abudurexiti, Zhang, Ting, Liu, Jia, Lu, Fengmin, Chen, Xiangmei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416172/
https://www.ncbi.nlm.nih.gov/pubmed/34485380
http://dx.doi.org/10.3389/fmolb.2021.676957
_version_ 1783748124428206080
author Chen, Chia-Chen
Guan, Guiwen
Qi, Xuewei
Abulaiti, Abudurexiti
Zhang, Ting
Liu, Jia
Lu, Fengmin
Chen, Xiangmei
author_facet Chen, Chia-Chen
Guan, Guiwen
Qi, Xuewei
Abulaiti, Abudurexiti
Zhang, Ting
Liu, Jia
Lu, Fengmin
Chen, Xiangmei
author_sort Chen, Chia-Chen
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8416172
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-84161722021-09-04 Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System Chen, Chia-Chen Guan, Guiwen Qi, Xuewei Abulaiti, Abudurexiti Zhang, Ting Liu, Jia Lu, Fengmin Chen, Xiangmei Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2021-08-17 /pmc/articles/PMC8416172/ /pubmed/34485380 http://dx.doi.org/10.3389/fmolb.2021.676957 Text en Copyright © 2021 Chen, Guan, Qi, Abulaiti, Zhang, Liu, Lu and Chen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Chen, Chia-Chen
Guan, Guiwen
Qi, Xuewei
Abulaiti, Abudurexiti
Zhang, Ting
Liu, Jia
Lu, Fengmin
Chen, Xiangmei
Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title_full Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title_fullStr Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title_full_unstemmed Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title_short Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System
title_sort pacbio sequencing of plc/prf/5 cell line and clearance of hbv integration through crispr/cas-9 system
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416172/
https://www.ncbi.nlm.nih.gov/pubmed/34485380
http://dx.doi.org/10.3389/fmolb.2021.676957
work_keys_str_mv AT chenchiachen pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT guanguiwen pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT qixuewei pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT abulaitiabudurexiti pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT zhangting pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT liujia pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT lufengmin pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system
AT chenxiangmei pacbiosequencingofplcprf5celllineandclearanceofhbvintegrationthroughcrisprcas9system