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

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-associ...

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Autores principales: Morgens, David W., Nandakumar, Divya, Didychuk, Allison L., Yang, Kevin J., Glaunsinger, Britt A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8797222/
https://www.ncbi.nlm.nih.gov/pubmed/35041709
http://dx.doi.org/10.1371/journal.ppat.1010236
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author Morgens, David W.
Nandakumar, Divya
Didychuk, Allison L.
Yang, Kevin J.
Glaunsinger, Britt A.
author_facet Morgens, David W.
Nandakumar, Divya
Didychuk, Allison L.
Yang, Kevin J.
Glaunsinger, Britt A.
author_sort Morgens, David W.
collection PubMed
description 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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spelling pubmed-87972222022-01-29 A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains Morgens, David W. Nandakumar, Divya Didychuk, Allison L. Yang, Kevin J. Glaunsinger, Britt A. PLoS Pathog Research Article 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. Public Library of Science 2022-01-18 /pmc/articles/PMC8797222/ /pubmed/35041709 http://dx.doi.org/10.1371/journal.ppat.1010236 Text en © 2022 Morgens et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Morgens, David W.
Nandakumar, Divya
Didychuk, Allison L.
Yang, Kevin J.
Glaunsinger, Britt A.
A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title_full A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title_fullStr A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title_full_unstemmed A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title_short A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
title_sort two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8797222/
https://www.ncbi.nlm.nih.gov/pubmed/35041709
http://dx.doi.org/10.1371/journal.ppat.1010236
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