Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells.

Chaolong Lin,Mengru Hao,Quan Yuan,Ningshao Xia,Chenghao Huang,Dan Xiong,Yong Luo,Huanhuan Li,Jun Zhang

doi:10.1038/srep34531

Abstract

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.

Highlights

Introduction of the non-homologous end joining (NHEJ) inhibitorSCR7 during CRISPR/Cas9-mediated editing of the HSV-1 genome.The previous data showed that a number of our Cas9-edited HSV-1 viruses were not GFP-positive, indicating that a substantial number of the viruses had been repaired by NHEJ rather than by homology-directed repair (HDR) with the donor plasmids
The efficiency of the CRISPR/Cas9-mediated cleavage was highest at a multiplicities of infections (MOIs) of between 0.1 and 1, and it decreased at a higher MOIs (5 or 10), likely due to the presence of excess uninfected virus
The results showed that the efficiency of the naturally occurring HDR was lower than 10−5; the efficiency of CRISPR/Cas9-mediated HDR of large gene was significantly increased to approximately 2.1%, which has never been reported before (Fig. 4b)

Summary

Introduction

The previous data showed that a number of our Cas9-edited HSV-1 viruses were not GFP-positive, indicating that a substantial number of the viruses had been repaired by NHEJ rather than by HDR with the donor plasmids. It was questioned whether introducing an NHEJ inhibitor could further improve the recombination efficiency of a desired recombinant virus. Introducing SCR7 into the CRISPR/Cas[9] system could greatly improve the gene replacement efficiency on the HSV-1 genome by inhibiting NHEJ-directed repair, which would facilitate the production of desired recombinant viruses (Fig. 5d)

Objectives

Methods

Results

Conclusion