Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells.
Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells.
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提高人类细胞中 HSV-1 病毒 CRISPR/Cas9 介导的精确基因组编辑的效率
DOI：
10.1038/srep34531
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发表时间：
2016-10-07
影响因子：
4.6
通讯作者：
Xia N
中科院分区：
文献类型：
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作者：
Lin C;Li H;Hao M;Xiong D;Luo Y;Huang C;Yuan Q;Zhang J;Xia N
基因修饰的单纯疱疹病毒1型（HSV - 1）是肿瘤治疗和疫苗研发的有前景的载体。CRISPR/Cas9系统是对生物体基因组进行精确基因编辑的最强大工具之一。然而，CRISPR/Cas9系统是否能在HSV - 1基因组中精确且高效地进行基因替换基本上仍不为人知。在此，我们报道了在人类细胞中CRISPR/Cas9介导的HSV - 1基因组编辑，包括大基因的敲除和替换。在稳定表达CRISPR/Cas9的已建立细胞中，引导RNA（gRNA）与Cas9协同作用，可在预先定义的靶区域内进行精确切割，并且通过同源定向修复（HDR）介导的基因替换，成功地利用外源基因无缝替换靶基因。将非同源末端连接（NHEJ）抑制剂SCR7引入CRISPR/Cas9系统极大地促进了HSV - 1基因组中HDR介导的基因替换。我们提供了首个遗传学证据，即同一HSV - 1基因组上不同位置的两个ICP0基因拷贝能够被高效地同时修饰，且无脱靶修饰。我们还开发了一种利用单细胞分选技术分离所需重组病毒的创新性平台。总之，我们的工作为DNA病毒的靶向编辑提供了一种显著改进的方法，这将促进抗癌溶瘤病毒和疫苗的开发。
Genetically modified HSV-1 viruses serve as promising vectors for tumour therapy and vaccine development. The CRISPR/Cas9 system is one of the most powerful tools for precise gene editing of the genomes of organisms. However, whether the CRISPR/Cas9 system can precisely and efficiently make gene replacements in the genome of HSV-1 remains essentially unknown. Here, we reported CRISPR/Cas9-mediated editing of the HSV-1 genome in human cells, including the knockout and replacement of large genes. In established cells stably expressing CRISPR/Cas9, gRNA in coordination with Cas9 could direct a precise cleavage within a pre-defined target region and foreign genes were successfully used to replace the target gene seamlessly by HDR-mediated gene replacement. Introducing the NHEJ inhibitor SCR7 to the CRISPR/Cas9 system greatly facilitated HDR-mediated gene replacement in the HSV-1 genome. We provided the first genetic evidence that two copies of the ICP0 gene in different locations on the same HSV-1 genome could be simultaneously modified with high efficiency and with no off-target modifications. We also developed a revolutionized isolation platform for desired recombinant viruses using single-cell sorting. Together, our work provides a significantly improved method for targeted editing of DNA viruses, which will facilitate the development of anti-cancer oncolytic viruses and vaccines.
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影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
RNA 引导核酸酶对大型病毒基因组进行高效靶向编辑
影响因子：
6.7
作者：
Bi Y;Sun L;Gao D;Ding C;Li Z;Li Y;Cun W;Li Q
通讯作者：
Li Q
影响因子：
46.9
作者：
Ghorbal, Mehdi;Gorman, Molly;Lopez-Rubio, Jose-Juan
通讯作者：
Lopez-Rubio, Jose-Juan
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao
影响因子：
46.9
作者：
通讯作者：
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