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.
Highlights
With the rapid development of biotechnology, site-specific genome editing approaches allow researchers to target any gene within any organism
At 24 hours posttransfection, the cells were infected with adenoviral vector (ADV)-Enhanced Green Fluorescent Protein (EGFP) at a multiplicity of infection (m.o.i.) of 1 PFU/cell, and the progeny viral genomes (P1) were harvested when cytopathic effects (CPEs) occurred
Each other type of mutation was found in one or two samples (Figure 1E). We examined whether such mutations could be passed down to progeny viruses by infecting cells with the P1 virus and extracting the progeny viral genomes (P2) when CPEs occurred
Summary
With the rapid development of biotechnology, site-specific genome editing approaches allow researchers to target any gene within any organism. Two well-known genome-editing technologies include zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These approaches function by using a nuclease to target a gene and cleave its DNA to induce double-stranded breaks (DSBs) at the target site. The Cas protein belongs to the type II CRISPR-Cas system that, with the guidance of a CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), cleaves DNA matching the crRNA in a sequence-specific manner [7,8]. A recent study by Jinek et al demonstrated that the crRNA-tracrRNA complex can be fused to form guide RNAs (gRNAs) that function in specific DNA recognition and Cas protein binding [8].
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