Engineered TALE-Fusion Proteins for Specific Gene Targeting
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Solution Overview
Problem
Current methods for regulating gene expression and genome editing, particularly using zinc finger nucleases and TALENs, face challenges in specificity and efficiency when targeting endogenous genes in higher eukaryotic cells, necessitating the development of more effective DNA binding domains and fusion proteins for therapeutic and diagnostic applications.
Innovation Solution
Engineered DNA-binding proteins comprising TALE-repeat units fused with functional domains, such as transcription factors, nucleases, and recombinases, are developed to form specific and efficient fusion proteins that can target and modify endogenous genes by recognizing unique DNA sequences, including those with atypical RVDs and varying gap spacings, enhancing their binding affinity and cleavage capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If zinc finger nucleases or TALENs are used for genome editing, then gene targeting capability is achieved, but specificity and efficiency when targeting endogenous genes in higher eukaryotic cells deteriorates
Solution Approach 1:
The DNA-binding domain is divided into modular TALE-repeat units, where each repeat unit specifically recognizes one DNA base pair. This segmentation allows for precise customization of the binding specificity by assembling different combinations of repeat units, thereby improving the ability to target specific endogenous genes in higher eukaryotic cells with enhanced specificity and efficiency
Solution Approach 2:
The patent employs atypical RVDs (Repeat Variable Diresidues) with altered amino acid compositions at positions 12 and 13 of the TALE-repeat units. This parameter change in the chemical structure of the DNA-binding domain enables recognition of non-canonical DNA sequences, expanding the targeting capability and improving specificity for endogenous genes that were previously difficult to target with conventional zinc finger or TALEN systems
2Reliability
If DNA binding proteins are engineered to recognize unique DNA sequences, then binding affinity is improved, but device complexity increases
Solution Approach 1:
The TALE-repeat units serve multiple functions: they provide DNA sequence recognition through their RVDs, maintain structural stability through their conserved hydrophobic core, and enable modular assembly for customized targeting. This multi-functionality within a standardized repeat unit framework allows the protein to achieve high binding affinity for unique DNA sequences without proportionally increasing overall structural complexity
Solution Approach 2:
The DNA-binding protein is constructed as a composite of standardized TALE-repeat modules, each with a uniform structural framework but variable RVD sequences. This composite architecture allows the protein to achieve high binding affinity through optimized sequence-specific interactions while maintaining relatively simple and predictable overall protein structure, facilitating easier engineering and characterization
3Adaptability or versatility
If TALE-repeat units with atypical RVDs are used, then DNA sequence recognition capability is improved, but manufacturing difficulty increases
Solution Approach 1:
The DNA-binding domain is segmented into standardized TALE-repeat units with atypical RVDs, where each unit can be independently designed and assembled. This segmentation allows for modular optimization of DNA sequence recognition capability while maintaining a uniform structural framework that simplifies manufacturing processes, as the same production protocols can be applied to different repeat unit combinations
Solution Approach 2:
The patent systematically varies the amino acid parameters at positions 12 and 13 of the TALE-repeat units to create atypical RVDs with enhanced or altered DNA sequence recognition capabilities. These controlled parameter changes are implemented within the conserved structural context of the TALE-repeat framework, allowing for improved adaptability to different DNA sequences while maintaining protein stability and manufacturability through standardized expression and purification protocols
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
These engineered TALE-fusion proteins enable precise regulation of gene expression and genomic modifications, offering improved specificity and activity for therapeutic applications, including genetic disease treatment, cancer research, and plant development, by effectively activating or repressing target genes and introducing specific mutations or sequences into the genome.
Implementation Method 1
Engineered DNA-binding proteins comprising TALE-repeat units fused with functional domains... that can target and modify endogenous genes by recognizing unique DNA sequences
Data Source
AI summary
Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.