Dataset Information

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

ABSTRACT: CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

SUBMITTER: Hunt C

PROVIDER: S-EPMC8119962 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

Hunt Charleen C Hartford Suzanne A SA White Derek D Pefanis Evangelos E Hanna Timothy T Herman Clarissa C Wiley Jarrell J Brown Heather H Su Qi Q Xin Yurong Y Voronin Dennis D Nguyen Hien H Altarejos Judith J Crosby Keith K Haines Jeffery J Cancelarich Sarah S Drummond Meghan M Moller-Tank Sven S Malpass Ryan R Buckley Jacqueline J Del Pilar Molina-Portela Maria M Droguett Gustavo G Frendewey David D Chiao Eric E Zambrowicz Brian B Gong Guochun G

Nature communications 20210513 1

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expre ...[more]

PMID: 33986266

Similar Datasets

Project description:Stem cell therapies have shown promise for regenerative treatment for musculoskeletal conditions, but their success is mixed. To enhance regenerative effects, growth factors are utilized to induce differentiation into native cell types, but uncontrollable in vivo conditions inhibit differentiation, and precise control of expressed matrix proteins is difficult to achieve. To address these issues, we investigated a novel method of enhancing regenerative phenotype through direct upregulation of major cartilaginous tissue proteins, aggrecan (ACAN), and collagen II (COL2A1) using dCas9-VPR CRISPR gene activation systems. We demonstrated increased expression and deposition of targeted proteins independent of exogenous growth factors in pellet culture. Singular upregulation of COL2A1/ACAN interestingly indicates that COL2A1 upregulation mediates the highest sulfated glycosaminoglycan (sGAG) deposition, in addition to collagen II deposition. Through RNA-seq analysis, this was shown to occur by COL2A1 upregulation mediating broader chondrogenic gene expression changes. Multiplex upregulation of COL2A1 and ACAN together resulted in the highest sGAG, and collagen II deposition, with levels comparable to those in chondrogenic growth factor-differentiated pellets. Overall, this work indicates dCas9-VPR systems can robustly upregulate COL2A1 and ACAN deposition without growth factors, to provide a novel, precise method of controlling stem cell phenotype for cartilage and intervertebral disc cell therapies and tissue engineering. Impact statement Stem cell therapies have come about as a potential regenerative treatment for musculoskeletal disease, but clinically, they have mixed results. To improve stem cell therapies, growth factors are used to aid a regenerative cell phenotype, but their effects are inhibited by in vivo musculoskeletal disease environments. This article describes CRISPR gene activation-based cell engineering methods that provide a growth factor-free method of inducing chondrogenic extracellular matrix deposition. This method is demonstrated to be as/more potent as growth factors in inducing a chondrogenic phenotype in pellet culture, indicating potential utility as a method of enhancing stem cell therapies for musculoskeletal disease.

Dataset Information

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

Publications

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets