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High-efficiency nonhomologous insertion of a foreign gene into the herpes simplex virus genome.

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机构: [1]Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China. [2]Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China. [3]The First Affiliated Hospital of Kunming Medical University, Kunming 650118, Yunnan, PR China.
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关键词: Herpes simplex virus CRISPR/Cas9 gene knock-in nonhomologous end joining

摘要:
Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.

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出版当年[2021]版:
大类 | 3 区 医学
小类 | 3 区 生物工程与应用微生物 3 区 病毒学
最新[2023]版:
大类 | 4 区 医学
小类 | 3 区 生物工程与应用微生物 4 区 病毒学
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出版当年[2020]版:
Q2 VIROLOGY Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
最新[2023]版:
Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Q2 VIROLOGY

影响因子: 最新[2023版] 最新五年平均 出版当年[2020版] 出版当年五年平均 出版前一年[2019版] 出版后一年[2021版]

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第一作者机构: [1]Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China. [2]Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China.
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