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-a...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-01-01
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| Series: | PLoS Pathogens |
| Online Access: | https://doi.org/10.1371/journal.ppat.1010236 |
| _version_ | 1818986935996121088 |
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| author | David W Morgens Divya Nandakumar Allison L Didychuk Kevin J Yang Britt A Glaunsinger |
| author_facet | David W Morgens Divya Nandakumar Allison L Didychuk Kevin J Yang Britt A Glaunsinger |
| author_sort | David W Morgens |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-20T18:58:42Z |
| format | Article |
| id | doaj.art-1ba31f7a603c449ebb7b9eb2917ecefc |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-12-20T18:58:42Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-1ba31f7a603c449ebb7b9eb2917ecefc2022-12-21T19:29:26ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742022-01-01181e101023610.1371/journal.ppat.1010236A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains.David W MorgensDivya NandakumarAllison L DidychukKevin J YangBritt A GlaunsingerWhile 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.https://doi.org/10.1371/journal.ppat.1010236 |
| spellingShingle | David W Morgens Divya Nandakumar Allison L Didychuk Kevin J Yang Britt A Glaunsinger A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains. PLoS Pathogens |
| 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 |
| url | https://doi.org/10.1371/journal.ppat.1010236 |
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