Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos
Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cel...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2025-07-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/87722 |
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| author | Oana Pelea Sarah Mayes Quentin RV Ferry Tudor A Fulga Tatjana Sauka-Spengler |
| author_facet | Oana Pelea Sarah Mayes Quentin RV Ferry Tudor A Fulga Tatjana Sauka-Spengler |
| author_sort | Oana Pelea |
| collection | DOAJ |
| description | Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo. Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers. |
| format | Article |
| id | doaj-art-eebc8e643f6d44d0a04b4325fe7898d1 |
| institution | Matheson Library |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-07-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-eebc8e643f6d44d0a04b4325fe7898d12025-07-29T14:00:45ZengeLife Sciences Publications LtdeLife2050-084X2025-07-011210.7554/eLife.87722Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryosOana Pelea0https://orcid.org/0000-0002-1968-9827Sarah Mayes1https://orcid.org/0000-0001-7630-9272Quentin RV Ferry2https://orcid.org/0000-0003-1843-8572Tudor A Fulga3https://orcid.org/0000-0002-1056-0082Tatjana Sauka-Spengler4https://orcid.org/0000-0001-9289-0263University of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, United KingdomUniversity of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, United KingdomUniversity of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, United KingdomUniversity of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, United KingdomUniversity of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, United Kingdom; Stowers Institute for Medical Research, Kansas City, United StatesCellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo. Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.https://elifesciences.org/articles/87722CRISPRconditional activationsynthetic biologyRNA biomarkers |
| spellingShingle | Oana Pelea Sarah Mayes Quentin RV Ferry Tudor A Fulga Tatjana Sauka-Spengler Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos eLife CRISPR conditional activation synthetic biology RNA biomarkers |
| title | Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos |
| title_full | Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos |
| title_fullStr | Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos |
| title_full_unstemmed | Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos |
| title_short | Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos |
| title_sort | specific modulation of crispr transcriptional activators through rna sensing guide rnas in mammalian cells and zebrafish embryos |
| topic | CRISPR conditional activation synthetic biology RNA biomarkers |
| url | https://elifesciences.org/articles/87722 |
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