Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

Arvidsson, Elin LU orcid ; Lobo, Diana Duarte ; Sabarese, Ermelinda LU ; Duarte, Fabio ; Nobre, Rui Jorge ; Quintino, Luis LU orcid and Lundberg, Cecilia LU orcid (2025) In Frontiers in Bioengineering and Biotechnology 13.
Abstract

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... (More)

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.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Adam17, CRISPRa, gene activation, gene therapy, Sirt1, Tfeb
in
Frontiers in Bioengineering and Biotechnology
volume
13
article number
1336313
publisher
Frontiers Media S. A.
external identifiers
  • scopus:85216919964
  • pmid:39917018
ISSN
2296-4185
DOI
10.3389/fbioe.2025.1336313
language
English
LU publication?
yes
additional info
Publisher Copyright: Copyright © 2025 Arvidsson, Lobo, Sabarese, Duarte, Nobre, Quintino and Lundberg.
id
ad2bb645-ac5f-45cc-ae0a-88db10387cc6
date added to LUP
2025-06-24 11:48:55
date last changed
2025-06-25 03:00:02
@article{ad2bb645-ac5f-45cc-ae0a-88db10387cc6,
  abstract     = {{<p>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.</p>}},
  author       = {{Arvidsson, Elin and Lobo, Diana Duarte and Sabarese, Ermelinda and Duarte, Fabio and Nobre, Rui Jorge and Quintino, Luis and Lundberg, Cecilia}},
  issn         = {{2296-4185}},
  keywords     = {{Adam17; CRISPRa; gene activation; gene therapy; Sirt1; Tfeb}},
  language     = {{eng}},
  publisher    = {{Frontiers Media S. A.}},
  series       = {{Frontiers in Bioengineering and Biotechnology}},
  title        = {{A systematic screening assay identifies efficient small guide RNAs for CRISPR activation}},
  url          = {{http://dx.doi.org/10.3389/fbioe.2025.1336313}},
  doi          = {{10.3389/fbioe.2025.1336313}},
  volume       = {{13}},
  year         = {{2025}},
}