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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...

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Autores principales: Martinez, Maria Guadalupe, Combe, Emmanuel, Inchauspe, Aurore, Mangeot, Philippe Emmanuel, Delberghe, Elodie, Chapus, Fleur, Neveu, Gregory, Alam, Antoine, Carter, Kara, Testoni, Barbara, Zoulim, Fabien
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
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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.
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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
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