Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity

Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million...

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Autores principales:	A. P. Kostyusheva, S. A. Brezgin, D. N. Zarifyan, D. S. Chistyakov, V. I. Gegechkory, E O. Bayurova, E. A. Volchkova, D. S. Kostyushev, V. P. Chulanov
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2019
Materias:	hepatitis b virus circular covalently closed dna crispr/cas9 specificity off-target effects efficiency modified grnas Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/a83a0ac4e8944c04897cf85ab0343c03
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spelling	oai:doaj.org-article:a83a0ac4e8944c04897cf85ab0343c032021-11-22T07:09:52ZHepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity2220-76192313-739810.15789/2220-7619-2019-2-279-287https://doaj.org/article/a83a0ac4e8944c04897cf85ab0343c032019-07-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/749https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs.A. P. KostyushevaS. A. BrezginD. N. ZarifyanD. S. ChistyakovV. I. GegechkoryE O. BayurovaE. A. VolchkovaD. S. KostyushevV. P. ChulanovSankt-Peterburg : NIIÈM imeni Pasteraarticlehepatitis b viruscircular covalently closed dnacrispr/cas9specificityoff-target effectsefficiencymodified grnasInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 9, Iss 2, Pp 279-287 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	hepatitis b virus circular covalently closed dna crispr/cas9 specificity off-target effects efficiency modified grnas Infectious and parasitic diseases RC109-216
spellingShingle	hepatitis b virus circular covalently closed dna crispr/cas9 specificity off-target effects efficiency modified grnas Infectious and parasitic diseases RC109-216 A. P. Kostyusheva S. A. Brezgin D. N. Zarifyan D. S. Chistyakov V. I. Gegechkory E O. Bayurova E. A. Volchkova D. S. Kostyushev V. P. Chulanov Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
description	Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs.
format	article
author	A. P. Kostyusheva S. A. Brezgin D. N. Zarifyan D. S. Chistyakov V. I. Gegechkory E O. Bayurova E. A. Volchkova D. S. Kostyushev V. P. Chulanov
author_facet	A. P. Kostyusheva S. A. Brezgin D. N. Zarifyan D. S. Chistyakov V. I. Gegechkory E O. Bayurova E. A. Volchkova D. S. Kostyushev V. P. Chulanov
author_sort	A. P. Kostyusheva
title	Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
title_short	Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
title_full	Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
title_fullStr	Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
title_full_unstemmed	Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity
title_sort	hepatitis b virus and site-specific nucleases: effects of genetic modifications in crispr/cas9 on antiviral activity
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2019
url	https://doaj.org/article/a83a0ac4e8944c04897cf85ab0343c03
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Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity

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