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PLoS Pathog. 2014 May 1;10(5):e1004090. doi: 10.1371/journal.ppat.1004090. eCollection 2014 May.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Author information

1
Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, Yunnan, China; Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, Yunnan, China.

Abstract

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

PMID:
24788700
PMCID:
PMC4006927
DOI:
10.1371/journal.ppat.1004090
[Indexed for MEDLINE]
Free PMC Article

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