TALE RVD Recognition for Multi-Gene Genome Editing
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Solution Overview
Problem
Existing genome editing techniques struggle to simultaneously modify multiple genes with similar sequences due to functional redundancy and nucleotide sequence variations, leading to inconsistent phenotypic outcomes.
Innovation Solution
A modified TALE system with tailored Repeat Variable Di-residue (RVD) combinations that recognize or tolerate nucleotides A, T, G, or C, allowing simultaneous editing of multiple genes with similar functions using a single enzyme.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional TALE with standard RVD combinations is used for genome editing, then high binding specificity to exact match sequences is achieved, but inability to simultaneously edit multiple genes with similar but not identical sequences occurs
Solution Approach 1:
The patent modifies the RVD amino acid combinations in the TALE repeat sequences to create new recognition specificities. By changing the chemical properties and binding characteristics of the RVDs, the system can tolerate certain nucleotide variations while maintaining sufficient binding strength, enabling simultaneous editing of multiple genes with similar sequences.
Solution Approach 2:
The patent designs TALE proteins with RVD combinations that can recognize multiple nucleotide variants (e.g., NT recognizing A or T, SD recognizing C or G). This multi-functionality allows a single TALE construct to bind to and edit multiple target genes that share high but not identical sequence similarity, achieving universal editing capability across gene families.
2Reliability
If TALE recognizes a sequence consisting of about 40 nucleotides, then off-target editing is reduced, but difficulty to edit multiple similar sequences simultaneously increases
Solution Approach 1:
The patent applies different RVD combinations at different positions within the TALE repeat array. By optimizing the local recognition properties at each position, the system achieves a balance between maintaining long-range binding specificity (reducing off-target effects) and accommodating local sequence variations (enabling multi-gene editing).
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
Enables efficient simultaneous modification of multiple genes with similar functions, even when their sequences are not completely identical, enhancing the precision and effectiveness of genome editing.
Implementation Method 1
The DNA-binding domain of TALE has a structure in which 10 to 30 amino acid repeat sequences each consisting of approximately 34 amino acid residues are arranged in tandem, and binds to a target nucleotide sequence on the genome
Implementation Method 2
an artificial endonuclease in which an endonuclease is linked to the DNA-binding domain of TALE can be used as a sequence-specific endonuclease, TALEN
Implementation Method 3
a fused body of TALE with cytidine deaminase (CD) or adenosine deaminase (ADA), which can modify double-stranded DNA, can be used to specifically modify a desired nucleotide
Implementation Method 4
a fused body of TALE with cytidine deaminase (CD) or adenosine deaminase (ADA), which can modify double-stranded DNA, can be used to specifically modify a desired nucleotide
Data Source
AI summary
It is an object of the present invention to provide a method for modifying multiple DNAs encoding identical or similar proteins using TALE, when the multiple DNAs are present. More specifically, the present invention relates to a method for modifying multiple DNAs encoding identical or similar proteins, wherein the method comprises allowing the TALE portion of one type of TALE-modifier complex comprising at least one repeat sequence containing RVD (repeat variable di-residue) composed of amino acids that recognize or tolerate N, V, H, D, B, R, Y, M, W, S or K, to bind to the binding regions of the multiple DNAs.