Система CRISPR/Cas9, экспрессируемая с плазмиды, подавляет репликацию вируса простого герпеса первого типа в культуре клеток Vero

Карпов, Д. С.; Карпов, В.Л.; Климова, Р. Р.; Demidova, N. A.; Кущ, А А

doi:10.1134/s0026898419010051

Cited by 12 publications

(2 citation statements)

References 35 publications

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“…The genomes of herpesviruses are double-stranded DNA that replicate in the nucleus and are thus targetable by CRISPR/Cas9. Indeed, CRISPR-based targeting of herpesviruses has been explored as a way to suppress infection (Wang and Quake, 2014; Roehm et al , 2016; van Diemen et al , 2016; Karpov et al , 2019; Tso, West and Wood, 2019; Walter and Verdin, 2020). CRISPR has also been used to make functional insertions and knockouts on the viral genome (Suenaga et al , 2014; Yuen et al , 2015; BeltCappellino et al , 2019) or to perform pooled functional screens (Hein and Weissman, 2019; Gabaev et al , 2020) in much the same way as on the mammalian genome.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

Morgens

Nandakumar

Didychuk

et al. 2021

Preprint

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While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV - one cut site approximately every 8 base pairs - enabling the pooled screening of the entire genome. We used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 - and more specifically its DNA binding domain - is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

Morgens

Nandakumar

Didychuk

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections

Najafi

Tan

Aghamiri

et al. 2022

Biomedicine & Pharmacotherapy

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A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains

et al. 2022

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While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi’s sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

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Система CRISPR/Cas9, экспрессируемая с плазмиды, подавляет репликацию вируса простого герпеса первого типа в культуре клеток Vero

Cited by 12 publications

References 35 publications

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections

A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains

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