dCas9 Histone Acetyltransferase Fusion for Regulatory Element Screening
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Solution Overview
Problem
Current screening technologies are unable to directly target and manipulate gene regulatory elements, which play a critical role in determining cell phenotype and disease susceptibility, due to off-target effects and limitations in probing epigenetic properties.
Innovation Solution
The development of a CRISPR/Cas9-based epigenomic editing system that uses a dCas9 fusion protein with histone acetyltransferase activity to directly modulate epigenetic structures at genomic regulatory elements, enabling high-throughput screening for regulatory element function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional screening technologies (small molecules, RNA interference) are used to inhibit protein function or block translation, then screening can be performed, but off-target effects occur and direct targeting of genomic regulatory elements is impossible
Solution Approach 1:
The patent uses dCas9 (catalytically inactive Cas9) as an intermediary tool that binds to genomic DNA through guide RNA without causing double-strand breaks. This intermediary approach allows specific targeting of regulatory elements while avoiding the harmful off-target effects associated with conventional screening methods that inhibit protein function or block translation.
Solution Approach 2:
The patent replaces the mechanical/chemical screening approaches (small molecules binding to proteins, RNA interference blocking translation) with a programmable DNA-binding system. The dCas9-gRNA complex provides sequence-specific DNA binding through complementary base pairing, substituting the indirect protein-inhibition mechanism with direct genomic element targeting.
2Productivity
If conventional screening technologies are used, then screening can be performed, but the function of gene regulatory elements cannot be directly probed
Solution Approach 1:
The dCas9 system serves multiple functions: it can bind to any genomic location guided by custom gRNAs, recruit various effector domains (transcriptional activators, repressors, epigenetic modifiers), and enable both activation and repression of gene expression. This universal platform allows direct probing of regulatory element functions across the genome, overcoming the limitations of conventional screening technologies.
Solution Approach 2:
The patent changes the fundamental parameter of targeting from indirect protein inhibition to direct DNA sequence recognition. By using programmable gRNAs that complement target DNA sequences, the system achieves precise localization to regulatory elements, enabling functional probing that was previously impossible with conventional screening approaches.
3Manufacturing precision
If CRISPR/Cas9 nuclease activity is used for genome editing, then genomic modifications can be achieved, but direct manipulation of epigenetic properties at regulatory elements is limited
Solution Approach 1:
The patent extracts the DNA-binding capability of Cas9 while removing its nuclease activity by introducing catalytic mutations (D10A and H840A). This creates dCas9, a DNA-binding protein without cutting activity, allowing manipulation of epigenetic properties at regulatory elements without causing DNA damage or off-target mutations associated with nuclease activity.
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
This approach allows for precise identification and characterization of regulatory elements responsible for gene expression, overcoming the limitations of conventional screening methods and enabling the mapping of regulatory elements across the genome.
Implementation Method 1
a dCas9 fusion protein with histone acetyltransferase activity to directly modulate epigenetic structures at genomic regulatory elements
Data Source
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AI summary
Disclosed herein are methods of using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based epigenomic editing systems for high-throughput screening of regulatory element function.