ArticleMolecular therapy. Nucleic acids2019
A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9.
Article in Molecular therapy. Nucleic acids, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed.
- Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to targetMolecular therapy. Nucleic acids · 2026Article
- CRISPR-dCas9 Activation of TSG-6 in MSCs Modulates the Cargo of MSC-Derived Extracellular Vesicles and Attenuates Inflammatory Responses in Human Intervertebral Disc Cells In Vitro.Cellular and molecular bioengineering · 2025Article
- NOS3 regulates angiogenic potential of human induced pluripotent stem cell-derived endothelial cells.Biochemistry and biophysics reports · 2024Article
- Harnessing the evolving CRISPR/Cas9 for precision oncology.Journal of translational medicine · 2024Review
- Development of a CRISPRi Human Retinal Pigmented Epithelium Model for Functional Study of Age-Related Macular Degeneration Genes.International journal of molecular sciences · 2023Article
- Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen GeneInternational journal of molecular sciences · 2022Article
- Review
- Methods forFrontiers in cell and developmental biology · 2021Article
- New Technologies to Study Functional Genomics of Age-Related Macular Degeneration.Frontiers in cell and developmental biology · 2020Review
- Dead Cas Systems: Types, Principles, and Applications.International journal of molecular sciences · 2019Review
- Generation of Leydig-like cells: approaches, characterization, and challenges.Asian journal of andrologyReview
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Authors and funding
9 authors.
Funding
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Abstract
Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.
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