Split dCas9 Fusion Protein via Inteins for Epigenetic Editing
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Solution Overview
Problem
Current methods lack effective means to modify the methylation status of target genes within live cells, which is crucial for therapeutic approaches to diseases related to altered gene expression.
Innovation Solution
An epigenetic editing system utilizing two separate expression cassettes encoding split proteins, specifically a transcription activator and an epigenetic modifier, which are fused via an intein system to form a functional protein that targets and modifies gene methylation status, guided by small guide RNAs to specific genomic sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a large-sized recombinant protein is expressed using a single expression cassette, then the epigenetic editing function is complete, but the expression efficiency and protein quality are insufficient
Solution Approach 1:
The large-sized recombinant protein is divided into two separate polypeptide chains (first polypeptide chain containing transcription activator and dCas9, second polypeptide chain containing epigenetic modifier), each expressed from separate expression cassettes. This segmentation enables efficient expression of each chain while maintaining the complete epigenetic editing function when both chains are present in the cell
Solution Approach 2:
An intein system is introduced as an intermediary mechanism to facilitate the functional assembly of the two separate polypeptide chains. The intein-mediated protein splicing or translational readthrough enables the two independently expressed chains to form a functional complex, resolving the contradiction between separate expression and functional completeness
2Reliability
If the dCas9 protein is used as a large scaffold for recruiting effectors, then the epigenetic editing capability is achieved, but the protein size becomes too large for effective expression
Solution Approach 1:
The dCas9 protein is split into two separate polypeptide chains, with each chain containing a portion of the dCas9 structure along with associated functional domains. This segmentation reduces the size of each individual protein component while preserving the overall epigenetic editing capability through the combined function of both chains
Solution Approach 2:
Different functional domains are distributed to different polypeptide chains based on their local requirements. The first chain contains transcription activator and dCas9 components for DNA binding and transcription activation, while the second chain contains the epigenetic modifier for methylation/demethylation activities. This local optimization enables efficient expression of each chain while maintaining complete functionality
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 system effectively modulates gene expression by altering methylation profiles, demonstrating potential in treating conditions like CDKL5 deficiency disorder by reactivating silenced alleles and increasing gene expression.
Implementation Method 1
utilizing the unique feature of an intein system to ultimately rejoin the two halves to form one larger fusion protein with the intein spliced out
Implementation Method 2
guided by small guide RNAs to specific genomic sequences
Implementation Method 3
Modifying the epigenetic profile and therefore regulating the expression of a disease-relevant genomic sequence
Data Source
AI summary
Disclosed herein are systems, compositions and methods for using a split dCas protein system to modify the epigenetic profile of a gene of interest. The systems, compositions, and methods are useful for modifying the epigenetic profile of a particular gene within a cell, based on the discovery that effective expression of a larger-sized recombinant protein can be successfully achieved using two separate expression cassettes each encoding a half of the protein fused with a half of an intein, utilizing the unique feature of an intein system to ultimately rejoin the two halves to form one larger fusion protein with the intein spliced out.