A systematic screening assay identifies efficient small guide RNAs for CRISPR activation
CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay c...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1336313/full |
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| _version_ | 1832590839920984064 |
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| author | Elin Arvidsson Diana Duarte Lobo Diana Duarte Lobo Ermelinda Sabarese Fabio Duarte Rui Jorge Nobre Rui Jorge Nobre Rui Jorge Nobre Luis Quintino Cecilia Lundberg |
| author_facet | Elin Arvidsson Diana Duarte Lobo Diana Duarte Lobo Ermelinda Sabarese Fabio Duarte Rui Jorge Nobre Rui Jorge Nobre Rui Jorge Nobre Luis Quintino Cecilia Lundberg |
| author_sort | Elin Arvidsson |
| collection | DOAJ |
| description | CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated viral vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, including Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA level. Correlation analysis of gene activation relative to sgRNA binding site distance to transcription start-site or nearby transcription factor binding sites failed to detect common characteristics influencing gene activation in the selected promoter regions. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications. |
| format | Article |
| id | doaj-art-b12bc81bfee047e8b216caad02ed00a8 |
| institution | Kabale University |
| issn | 2296-4185 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-b12bc81bfee047e8b216caad02ed00a82025-01-23T06:56:36ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852025-01-011310.3389/fbioe.2025.13363131336313A systematic screening assay identifies efficient small guide RNAs for CRISPR activationElin Arvidsson0Diana Duarte Lobo1Diana Duarte Lobo2Ermelinda Sabarese3Fabio Duarte4Rui Jorge Nobre5Rui Jorge Nobre6Rui Jorge Nobre7Luis Quintino8Cecilia Lundberg9CNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, SwedenCNC - Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, PortugalInstitute for Interdisciplinary Research, University of Coimbra, Coimbra, PortugalCNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, SwedenCNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, SwedenCNC - Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, Coimbra, PortugalInstitute for Interdisciplinary Research, University of Coimbra, Coimbra, PortugalViraVector – Viral Vector for Gene Transfer Core Facility, University of Coimbra, Coimbra, PortugalCNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, SwedenCNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, SwedenCRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated viral vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, including Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA level. Correlation analysis of gene activation relative to sgRNA binding site distance to transcription start-site or nearby transcription factor binding sites failed to detect common characteristics influencing gene activation in the selected promoter regions. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.https://www.frontiersin.org/articles/10.3389/fbioe.2025.1336313/fullCRISPRagene activationgene therapyTfebAdam17Sirt1 |
| spellingShingle | Elin Arvidsson Diana Duarte Lobo Diana Duarte Lobo Ermelinda Sabarese Fabio Duarte Rui Jorge Nobre Rui Jorge Nobre Rui Jorge Nobre Luis Quintino Cecilia Lundberg A systematic screening assay identifies efficient small guide RNAs for CRISPR activation Frontiers in Bioengineering and Biotechnology CRISPRa gene activation gene therapy Tfeb Adam17 Sirt1 |
| title | A systematic screening assay identifies efficient small guide RNAs for CRISPR activation |
| title_full | A systematic screening assay identifies efficient small guide RNAs for CRISPR activation |
| title_fullStr | A systematic screening assay identifies efficient small guide RNAs for CRISPR activation |
| title_full_unstemmed | A systematic screening assay identifies efficient small guide RNAs for CRISPR activation |
| title_short | A systematic screening assay identifies efficient small guide RNAs for CRISPR activation |
| title_sort | systematic screening assay identifies efficient small guide rnas for crispr activation |
| topic | CRISPRa gene activation gene therapy Tfeb Adam17 Sirt1 |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1336313/full |
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