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ORIGINAL RESEARCH article

Front. Bioeng. Biotechnol.
Sec. Cell and Gene Therapy
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1336313

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

Provisionally accepted
Elin Arvidsson Elin Arvidsson 1Diana Duarte Lobo Diana Duarte Lobo 2Ermelinda Sabarese Ermelinda Sabarese 1Fabio Duarte Fabio Duarte 1Rui Jorge Nobre Rui Jorge Nobre 2Cecilia Lundberg Cecilia Lundberg 1*Luis Quintino Luis Quintino 1
  • 1 Lund University, Lund, Skane County, Sweden
  • 2 University of Coimbra, Coimbra, Coimbra, Portugal

The final, formatted version of the article will be published soon.

    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. 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.

    Keywords: CRISPRa, Gene Activation, Gene Therapy, TFEB, ADAM17, SIRT1

    Received: 10 Nov 2023; Accepted: 06 Jan 2025.

    Copyright: © 2025 Arvidsson, Lobo, Sabarese, Duarte, Nobre, Lundberg and Quintino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Cecilia Lundberg, Lund University, Lund, 221 00, Skane County, Sweden

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.