Human-Derived Transcription Activators for CRISPR Gene Regulation
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Solution Overview
Problem
Current CRISPR-based transcriptional activation systems face challenges due to large and restrictive transactivation components, reliance on viral-derived components that are poorly tolerated in clinically important cell types, and an underutilized repertoire of human transcription factors and chromatin modifiers.
Innovation Solution
Development of recombinant transcription activators comprising transcription activation domains from MRTF-A, STAT1, and eNRF2, which can be combined with genomic regulatory element targeting domains and RNA-binding proteins to enhance specificity and efficiency of gene regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Power
If viral-derived transactivation modules are used to achieve high transactivation potency, then transcriptional activation capability is improved, but cellular tolerance and clinical applicability deteriorate
Solution Approach 1:
The patent changes the source parameter of transactivation domains from viral pathogens to human proteins (MRTF-A, STAT1, eNRF2), fundamentally altering the biological compatibility parameter while maintaining transactivation function through systematic testing and optimization of human-derived components
Solution Approach 2:
The patent creates synthetic transactivation effectors by copying and combining functional domains from human transcription factors and chromatin modifiers, producing artificial but biocompatible transactivation modules that replicate the function of natural human proteins without the harmful attributes of viral-derived systems
2Power
If large transactivation components are used to achieve high transactivation potency, then transcriptional activation capability is improved, but deliverability and system compactness deteriorate
Solution Approach 1:
The patent segments the transactivation function into discrete, modular domains from human proteins (MRTF-A, STAT1, eNRF2), each contributing specific transactivation activities. These segmented domains can be组合 in various configurations to achieve desired transactivation potency while minimizing overall component size for better deliverability
Solution Approach 2:
The patent creates multi-functional transactivation effectors where a single effector molecule can perform multiple transactivation functions through the combination of different human protein domains, reducing the need for multiple separate large components and achieving compactness without sacrificing functional capability
Data Source
AI summary
The present disclosure is directed to designed fusion proteins derived from MTFs with strong potency to modulate transcription and designated these recombinant fusion proteins MSN and NMS. These powerful transactivators potently activate transcription from endogenous loci when recruited through CRISPR-dCas9, Zinc Finger, or TALE system proteins. This technology permits upregulation of gene expression in targeted manner devoid of viral transcription activation domains and is amenable to high-throughput screening. These synthetic transcription activators interact with all programable DNA binding proteins tested and have exhibited applicability in vitro for efficient lineage conversion.