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Related Concept Videos

CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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A systematic screening assay identifies efficient small guide RNAs for CRISPR activation.

Elin Arvidsson1, Diana Duarte Lobo2,3, Ermelinda Sabarese1

  • 1CNS Gene Therapy, Department of Experimental Medical Sciences, Lund University, Lund, Sweden.

Frontiers in Bioengineering and Biotechnology
|February 7, 2025
PubMed
Summary

This study introduces a new screening assay to identify effective single-guide RNAs (sgRNAs) for CRISPR-mediated gene activation (CRISPRa). The assay streamlines viral vector construction for gene therapy applications.

Keywords:
Adam17CRISPRaSirt1Tfebgene activationgene therapy

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Area of Science:

  • Biotechnology
  • Gene Therapy
  • Molecular Biology

Background:

  • CRISPR-mediated gene activation (CRISPRa) is a rapidly advancing field with significant therapeutic potential.
  • Efficient selection of single-guide RNAs (sgRNAs) is crucial for successful CRISPRa applications, but experimental validation tools are lacking.

Purpose of the Study:

  • To develop and validate a screening assay for identifying efficient sgRNAs for CRISPRa.
  • To establish a streamlined cloning workflow for incorporating selected sgRNAs into viral vectors.
  • To assess the performance of the workflow using therapeutically relevant genes.

Main Methods:

  • Development of an in vitro fluorescence-based screening assay for sgRNA efficiency.
  • Implementation of a Golden Gate cloning workflow for lentiviral (LVs) and adeno-associated viral vectors (AAVs).
  • Validation using neurodegenerative disease-related genes (Tfeb, Adam17, Sirt1).

Main Results:

  • The screening assay successfully identified efficient single- and double sgRNAs.
  • Selected sgRNAs demonstrated endogenous gene activation at the mRNA level.
  • Correlation analysis did not reveal common characteristics influencing gene activation based on binding site proximity.

Conclusions:

  • The developed screening assay and cloning workflow can identify functional sgRNA candidates for CRISPRa.
  • This approach can accelerate the development and application of CRISPRa-based gene therapies.
  • Further research is needed to understand the determinants of sgRNA efficiency in different genomic contexts.