tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9
Abstract CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Su...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-09-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-40836-3 |
| _version_ | 1797209293611597824 |
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| author | Ankush Auradkar Annabel Guichard Saluja Kaduwal Marketta Sneider Ethan Bier |
| author_facet | Ankush Auradkar Annabel Guichard Saluja Kaduwal Marketta Sneider Ethan Bier |
| author_sort | Ankush Auradkar |
| collection | DOAJ |
| description | Abstract CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14–15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression. |
| first_indexed | 2024-03-10T17:27:35Z |
| format | Article |
| id | doaj.art-913069c903b94379a10027ebc9b198a9 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-24T09:52:24Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-913069c903b94379a10027ebc9b198a92024-04-14T11:20:16ZengNature PortfolioNature Communications2041-17232023-09-0114111410.1038/s41467-023-40836-3tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9Ankush Auradkar0Annabel Guichard1Saluja Kaduwal2Marketta Sneider3Ethan Bier4Department of Cell and Developmental Biology, University of California, San DiegoDepartment of Cell and Developmental Biology, University of California, San DiegoDepartment of Cell and Developmental Biology, University of California, San DiegoDepartment of Cell and Developmental Biology, University of California, San DiegoDepartment of Cell and Developmental Biology, University of California, San DiegoAbstract CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14–15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.https://doi.org/10.1038/s41467-023-40836-3 |
| spellingShingle | Ankush Auradkar Annabel Guichard Saluja Kaduwal Marketta Sneider Ethan Bier tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 Nature Communications |
| title | tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 |
| title_full | tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 |
| title_fullStr | tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 |
| title_full_unstemmed | tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 |
| title_short | tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9 |
| title_sort | tgcrispri efficient gene knock down using truncated grnas and catalytically active cas9 |
| url | https://doi.org/10.1038/s41467-023-40836-3 |
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