对HBV cccDNA的CRISPR / Cas9诱发突变的整个光谱

StephenW

Mol Ther. 2016 May 16. doi: 10.1038/mt.2016.94. [Epub ahead of print]
Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA.Seeger C1, Sohn JA1.
Author information

• 1Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111.
  

AbstractHepatitis B virus causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in non-dividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV (Seeger and Sohn, 2014). Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA (Lucifora et al. 2014). In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by non-homologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.Molecular Therapy (2016); doi:10.1038/mt.2016.94.

StephenW

分子疗法。 2016年5月16日DOI：10.1038 / mt.2016.94。 [打印EPUB提前]
对HBV cccDNA的CRISPR / Cas9诱发突变的整个光谱。
西格C1，孙某JA1。
作者信息

    1Institute癌症研究，Fox Chase癌症中心，333 Cottman大道，费城，PA 19111。

抽象

乙肝病毒会导致还不能治愈慢性感染。该病毒仍然存在于感染的肝细胞，因为共价闭合环状DNA（cccDNA的），对病毒RNA的转录的模板，是在非分裂细胞稳定。与核苷类似物的抗病毒疗法抑制在感染的肝细胞的细胞质中衣壳HBV DNA的合成，但不破坏核的cccDNA。因为超过200万人感染仍然为慢性乙肝治愈（CHB）已成为抗病毒治疗的主要挑战之一。作为朝向治疗疗法的发展的第一步，我们以前表明，在CRISPR / Cas9系统可用于在功能上灭活从感染的HBV（西格和索恩2014）衍生的cccDNA。此外，一些证据表明，某些细胞因子可能诱发APOBEC介导的级联反应，导致cccDNA的破坏（Lucifora等，2014）。在这份报告中，我们研究了两种机制的结合是否能够协同作用灭活cccDNA的。使用下一代测序（NGS），我们确定了突变的cccDNA的以下Cas9切割和修通过非同源末端连接（NHEJ）的完整光谱。我们发现，HBV-DNA的90％以上是由Cas9切割。另外，我们的结果表明，Cas9切割后的HBV DNA的编辑是至少15000倍更高效，APOBEC介导的胞嘧啶脱氨处理后感染的细胞与干扰素α（IFNα）组成。我们还发现，先前使用的方法检测胞嘧啶脱氨基的DNA，称为3D-PCR高估的量和编辑的HBV DNA的频率。总之，我们的研究结果表明，CRISPR / Cas9系统是迄今为止在功能上灭活乙肝病毒的cccDNA并为CHB.Molecular治疗提供治愈（2016年）的最佳方法; DOI：10.1038 / mt.2016.94。

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