A CRISPR-based instant DNA repositioning system and the early intranuclear life of HSV-1
Authors: Fan Zhaoyang , Xiang Juan , Xu Pei 
Published: April 2022

AbstractThe intranuclear localization of viral DNA genomes in relation to the intranuclear environment plays critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of the infection, manipulative tools are needed. Instant repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to thenuclearperipheryinstantly (CRISPR-nuPin). Based on this system, investigations of herpes simplex virus 1 (HSV-1), a prototype member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: I) Viral genomes tethered to the nuclear periphery upon entry, compared with those in the nuclear interior, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent inhibition. II) Relocating HSV-1 genomes at 1 hour post infection significantly promoted transcription of viral β and γ genes, termed an “Escaping” effect. III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating “Escaping”. IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic should be widely applicable to many DNA viruses.SummaryThe intranuclear localization of viral DNA genomes plays a critical role in determining virus fate. To elucidate the causal relationship between subnuclear localization and the pathogenic outcome of DNA viruses, manipulative tools are needed. Herein, we report an inducible CRISPR-based two-component platform that relocates DNA pieces (5 kb to 170 kb) to thenuclearperipheryinstantly (CRISPR-nuPin). Utilizing this tactic, we interrogated the early intranuclear life of herpes simplex virus 1 (HSV-1), a prototype of human herpesviruses, in space and time and revealed that I) viral genomes tethered to the nuclear edge upon entry were prone to suppressive histone packaging and severe inhibition. II) Relocating HSV-1 genomes to the nuclear fringe at 1 hour post infection promoted transcription of viral genes (“Escaping”). III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating “Escaping”. IV) Subnuclear localization was a critical factor only during early infection.HighlightsCRISPR-nuPin is an inducible two-component DNA repositioning systemIt mediates instant nuclear edging of viral DNA during infectionA powerful approach to interrogate DNA viruses in space and timeViral DNA at the nuclear periphery upon entry is strongly silencedIn briefAn inducible two-component CRISPR-based platform that instantly repositions HSV-1 genomes to the nuclear edge unveils intranuclear space heterogeneity for the incoming viral genomes and dynamic stages of the host-virus interplay during early infection of the pathogen.