CRISPR SAM Animal Models for In Vivo Gene Activation Testing
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Solution Overview
Problem
Current methods for assessing the efficiency of CRISPR/Cas agents in vivo are limited by the need to transiently transfect DNA constructs, leading to potential toxic effects and difficulties in delivering biologically active agents to target cells or tissues, necessitating higher concentrations and increased risks.
Innovation Solution
Development of non-human animal models with genomically integrated CRISPR/Cas synergistic activation mediator (SAM) systems, comprising chimeric Cas proteins and guide RNAs, to efficiently activate transcription of target genes in vivo, using AAV-mediated delivery and tissue-specific recombinase systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transient transfection of DNA constructs is used to introduce CRISPR/Cas components, then the components can be delivered to cells, but the cells must undergo transcription and translation before Cas protein is available, increasing time and potential toxic effects
Solution Approach 1:
The patent introduces CRISPR/Cas components directly into the animal model's genome or cells in advance, so that the Cas protein is immediately available for interaction with sgRNA without requiring transient transcription and translation. This preliminary integration eliminates the time delay inherent in plasmid-based transient transfection methods.
2Reliability
If higher concentrations of CRISPR/Cas agents are used to overcome delivery difficulties, then more agents reach target cells, but the risk of toxic effects and side effects increases
Solution Approach 1:
The patent employs an intermediary delivery system (such as viral vectors or engineered particles) that facilitates the transport of CRISPR/Cas components to target cells. This intermediary mechanism improves delivery efficiency, allowing lower concentrations of agents to reach target cells effectively, thereby reducing toxic effects and side effects.
3Ease of manufacture
If transient transfection methods are used, then CRISPR/Cas components can be introduced, but the binding specificity of artificial transcription factors is degenerate and difficult to predict
Solution Approach 1:
The patent replaces the plasmid-based transient transfection system with a more precise delivery mechanism, such as genome integration or direct injection of functional components. This substitution enables better control over the expression and binding specificity of transcription factors, moving from degenerate and unpredictable binding to more precise and controllable interactions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enhances the assessment of CRISPR/Cas agent activity and delivery, allowing precise gene expression modulation with reduced toxic effects and improved targeting, achieving up to 20-fold higher expression levels compared to control animals.
Implementation Method 1
CRISPR/Cas technology is a promising new therapeutic modality and can be used not only to make targeted genomic modifications but to regulate transcription of target genes
Implementation Method 2
delivery of biologically active agents such as CRISPR/Cas agents to subjects is often hindered by difficulties in the components reaching the target cell or tissue
Data Source
AI summary
Non-human animal cells and non-human animals comprising CRISPR/Cas synergistic activation mediator system components and methods of making and using such non-human animal cells and non-human animals are provided. Methods are provided for using such non-human animals to increase expression of target genes in vivo and to assess CRISPR/Cas synergistic activation mediator systems for the ability to increase expression of target genes in vivo.