Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China.
Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China.
J Gen Virol. 2020 Sep;101(9):982-996. doi: 10.1099/jgv.0.001451.
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.
高效、准确、便捷的外源基因插入策略对于高通量、快速构建大型 DNA 病毒载体至关重要，但相对低效和劳动密集型的方法限制了重组病毒的应用。在本研究中，我们应用了基于非同源末端连接（NHEJ）修复途径的非同源插入（NHI）策略。与目前使用的同源重组（HR）策略相比，我们优化后获得了更高的外源基因插入单纯疱疹病毒（HSV）基因组的效率，达到 45%。通过使用 NHI，我们使用少量临床病毒快速构建了重组报告病毒，重组病毒至少稳定传代十次。根据测序结果，NHI 的保真度为 70-100%，与插入位点的序列背景有关。最后，我们通过定量脉冲分析描绘了 Cas9 快速切割外源基因供体质粒和病毒基因组的动态过程。此外，NHI 策略对野生型和反向整合病毒基因组施加选择压力，以有效地将外源基因按预定方向整合。我们的结果表明，合理设计的 NHI 策略可用于快速高效地将外源基因敲入 HSV 基因组，并为使用 NHI 将外源基因插入大型 DNA 病毒基因组提供有用的指导。
