1887

Abstract

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.

Funding
This study was supported by the:
  • Talent development project (Award D2017003)
  • National Natural Science Foundation of China, http://dx.doi.org/10.13039/501100001809 (Award 81971947)
  • Major Science and Technology Projects of Yunnan Province (Award 2019ZF001)
  • National Science and Technology Major Projects (Award 2018ZX09738003-007)
  • CAMS Initiative for Innovative Medicine (Award 2016-I2M-1-019)
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2020-06-30
2024-03-29
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