Cargando…
CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants
Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) d...
Autores principales: | , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9040760/ https://www.ncbi.nlm.nih.gov/pubmed/35389262 http://dx.doi.org/10.1128/mbio.02888-21 |
_version_ | 1784694403754885120 |
---|---|
author | Martinez, Maria Guadalupe Combe, Emmanuel Inchauspe, Aurore Mangeot, Philippe Emmanuel Delberghe, Elodie Chapus, Fleur Neveu, Gregory Alam, Antoine Carter, Kara Testoni, Barbara Zoulim, Fabien |
author_facet | Martinez, Maria Guadalupe Combe, Emmanuel Inchauspe, Aurore Mangeot, Philippe Emmanuel Delberghe, Elodie Chapus, Fleur Neveu, Gregory Alam, Antoine Carter, Kara Testoni, Barbara Zoulim, Fabien |
author_sort | Martinez, Maria Guadalupe |
collection | PubMed |
description | Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. |
format | Online Article Text |
id | pubmed-9040760 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-90407602022-04-27 CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants Martinez, Maria Guadalupe Combe, Emmanuel Inchauspe, Aurore Mangeot, Philippe Emmanuel Delberghe, Elodie Chapus, Fleur Neveu, Gregory Alam, Antoine Carter, Kara Testoni, Barbara Zoulim, Fabien mBio Research Article Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference. American Society for Microbiology 2022-04-07 /pmc/articles/PMC9040760/ /pubmed/35389262 http://dx.doi.org/10.1128/mbio.02888-21 Text en Copyright © 2022 Martinez et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Martinez, Maria Guadalupe Combe, Emmanuel Inchauspe, Aurore Mangeot, Philippe Emmanuel Delberghe, Elodie Chapus, Fleur Neveu, Gregory Alam, Antoine Carter, Kara Testoni, Barbara Zoulim, Fabien CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title | CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title_full | CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title_fullStr | CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title_full_unstemmed | CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title_short | CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants |
title_sort | crispr-cas9 targeting of hepatitis b virus covalently closed circular dna generates transcriptionally active episomal variants |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9040760/ https://www.ncbi.nlm.nih.gov/pubmed/35389262 http://dx.doi.org/10.1128/mbio.02888-21 |
work_keys_str_mv | AT martinezmariaguadalupe crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT combeemmanuel crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT inchauspeaurore crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT mangeotphilippeemmanuel crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT delbergheelodie crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT chapusfleur crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT neveugregory crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT alamantoine crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT carterkara crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT testonibarbara crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants AT zoulimfabien crisprcas9targetingofhepatitisbviruscovalentlyclosedcirculardnageneratestranscriptionallyactiveepisomalvariants |