直系同源CRISPR / Cas9系统用于特异性和有效降解乙型肝炎病毒

StephenW

Cell Mol Life Sci. 2019 Jan 23. doi: 10.1007/s00018-019-03021-8. [Epub ahead of print]
Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus.
Kostyushev D1, Brezgin S2,3, Kostyusheva A2, Zarifyan D2, Goptar I2,4, Chulanov V2,5.
Author information

1
    Viral Hepatitis Laboratory, Central Research Institute of Epidemiology, 3A Novogireevskaya Street, Moscow, 111123, Russian Federation. [email protected].
2
    Viral Hepatitis Laboratory, Central Research Institute of Epidemiology, 3A Novogireevskaya Street, Moscow, 111123, Russian Federation.
3
    Institute of Immunology, Federal Medical Biological Agency, Moscow, 115478, Russian Federation.
4
    Izmerov Research Institute of Occupational Health, Moscow, 105275, Russian Federation.
5
    Sechenov University, Moscow, 119146, Russian Federation.

Abstract

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.
KEYWORDS:

Antiviral; Cure; Liver; Mutations; NHEJ; Therapeutics

PMID:
    30673820
DOI:
    10.1007/s00018-019-03021-8

StephenW

Cell Mol Life Sci。 2019年1月23日doi：10.1007 / s00018-019-03021-8。 [印刷前的电子版]
直系同源CRISPR / Cas9系统用于特异性和有效降解乙型肝炎病毒的共价闭合环状DNA。
Kostyushev D1，Brezgin S2,3，Kostyusheva A2，Zarifyan D2，Goptar I2,4，Chulanov V2,5。
作者信息

1
    病毒性肝炎实验室，中央流行病学研究所，3 Novogireevskaya Street，Moscow，111123，Russian Federation。 [email protected]。
2
    病毒性肝炎实验室，中央流行病学研究所，3 Novogireevskaya Street，Moscow，111123，Russian Federation。
3
    联邦医学生物机构免疫学研究所，莫斯科，115478，俄罗斯联邦。
4
    Izmerov职业卫生研究所，莫斯科，105275，俄罗斯联邦。
五
    Sechenov大学，莫斯科，119146，俄罗斯联邦。

抽象

乙型肝炎病毒（HBV）的共价闭合环状DNA（cccDNA）是病毒持续存在和慢性乙型肝炎的主要原因.CRISPR / Cas9核酸酶可以特异性靶向HBV cccDNA进行衰变，但核酸酶在人类基因组限制中的脱靶效应他们的临床效用。来自四种不同物种的CRISPR / Cas9系统在细胞系中共表达，其中引导RNA靶向HBV基因组的保守区域。来自化脓性链球菌（Sp）和嗜热链球菌（St）的CRISPR / Cas9系统靶向HBV基因组的保守区域阻断HBV复制，并且最重要的是，在转染后6天导致超过90％的HBV cccDNA降解。通过抑制非同源末端连接途径损害HBV cccDNA的降解并导致HBV cccDNA的错误修复。 HBV cccDNA甲基化也影响CRISPR / Cas9的抗病毒活性。单核苷酸HBV遗传变异不影响St CRISPR / Cas9的抗HBV活性，表明其可用于靶向许多HBV变体。然而，两个或更多个错配损害或阻断CRISPR / Cas9活性，表明宿主DNA不可能被靶向。深度测序显示Sp CRISPR / Cas9诱导了脱靶诱变，而St CRISPR / Cas9对宿主基因组没有影响。 St CRISPR / Cas9系统是最安全的抗HBV活性系统。
关键词：

抗病毒;治愈;肝;突变; NHEJ;疗法

结论：
    30673820
DOI：
    10.1007 / s00018-019-03021-8

灵魂不屈

感谢分享！

齐欢畅

齐欢畅

深度测序显示Sp CRISPR / Cas9诱导了脱靶诱变，而St CRISPR / Cas9对宿主基因组没有影响。 St CRISPR / Cas9系统是最安全的抗HBV活性系统。

newchinabok

人类一定能攻克hbv  ，但是需要漫长的时间