Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing
Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitabl...
Main Authors: | , , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Elsevier
2020-06-01
|
Series: | Molecular Therapy: Nucleic Acids |
Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253120300962 |
id |
doaj-dd9b1aae44ad482ba469310eb3a054bf |
---|---|
record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yu-Chan Yang Yu-Hsiang Chen Jia-Horng Kao Chi Ching I-Jung Liu Chih-Chiang Wang Cheng-Hsueh Tsai Fang-Yi Wu Chun-Jen Liu Pei-Jer Chen Ding-Shinn Chen Hung-Chih Yang |
spellingShingle |
Yu-Chan Yang Yu-Hsiang Chen Jia-Horng Kao Chi Ching I-Jung Liu Chih-Chiang Wang Cheng-Hsueh Tsai Fang-Yi Wu Chun-Jen Liu Pei-Jer Chen Ding-Shinn Chen Hung-Chih Yang Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing Molecular Therapy: Nucleic Acids |
author_facet |
Yu-Chan Yang Yu-Hsiang Chen Jia-Horng Kao Chi Ching I-Jung Liu Chih-Chiang Wang Cheng-Hsueh Tsai Fang-Yi Wu Chun-Jen Liu Pei-Jer Chen Ding-Shinn Chen Hung-Chih Yang |
author_sort |
Yu-Chan Yang |
title |
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing |
title_short |
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing |
title_full |
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing |
title_fullStr |
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing |
title_full_unstemmed |
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing |
title_sort |
permanent inactivation of hbv genomes by crispr/cas9-mediated non-cleavage base editing |
publisher |
Elsevier |
series |
Molecular Therapy: Nucleic Acids |
issn |
2162-2531 |
publishDate |
2020-06-01 |
description |
Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated “base editors” (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome. |
url |
http://www.sciencedirect.com/science/article/pii/S2162253120300962 |
work_keys_str_mv |
AT yuchanyang permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT yuhsiangchen permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT jiahorngkao permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT chiching permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT ijungliu permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT chihchiangwang permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT chenghsuehtsai permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT fangyiwu permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT chunjenliu permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT peijerchen permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT dingshinnchen permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting AT hungchihyang permanentinactivationofhbvgenomesbycrisprcas9mediatednoncleavagebaseediting |
_version_ |
1724662053119983616 |
spelling |
doaj-dd9b1aae44ad482ba469310eb3a054bf2020-11-25T03:09:32ZengElsevierMolecular Therapy: Nucleic Acids2162-25312020-06-0120480490Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base EditingYu-Chan Yang0Yu-Hsiang Chen1Jia-Horng Kao2Chi Ching3I-Jung Liu4Chih-Chiang Wang5Cheng-Hsueh Tsai6Fang-Yi Wu7Chun-Jen Liu8Pei-Jer Chen9Ding-Shinn Chen10Hung-Chih Yang11Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanDepartment of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanDepartment of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanDepartment of Nursing, Cardinal Tien Junior College of Healthcare and Management, New Taipei City, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanDepartment of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanGraduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, TaiwanDepartment of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Corresponding author: Hung-Chih Yang, MD, PhD, Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Basic Medical Sciences Building, Room 724, 1, Jen-Ai Road, Section 1, Taipei 10051, Taiwan.Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated “base editors” (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.http://www.sciencedirect.com/science/article/pii/S2162253120300962 |