TALEN Specificity via Terminal Charge Reduction
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Solution Overview
Problem
Transcription activator-like effector nucleases (TALENs) face challenges in distinguishing target sequences from closely related off-target sequences, leading to undesired off-target cleavage, which is not well understood and affects their specificity and therapeutic applications.
Innovation Solution
Engineered TALENs with modified net charge and binding energy, specifically by replacing cationic amino acids with uncharged or negatively charged residues in the N-terminal and C-terminal domains, are developed to reduce non-specific DNA binding and enhance on-target cleavage specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional TALENs are used to target DNA sequences, then they can bind and cleave the target sequence, but they also exhibit off-target cleavage activity that reduces specificity
Solution Approach 1:
The patent modifies the net charge parameter of the TALEN protein by replacing cationic amino acids with uncharged or negatively charged residues in the N-terminal and C-terminal domains. This parameter change reduces non-specific electrostatic interactions with DNA, thereby minimizing off-target cleavage while preserving on-target activity. The specificity improvement of up to 116-fold demonstrates the effectiveness of this parameter modification approach.
2Measurement precision
If TALENs are designed to recognize specific DNA sequences, then they can perform targeted gene manipulation, but the ability to distinguish target from off-target sequences is not well understood
Solution Approach 1:
The patent systematically modifies the charge distribution parameters of TALEN domains to optimize sequence discrimination. By replacing cationic residues (lysine and arginine) with uncharged (glutamine, glycine) or negatively charged residues, the invention creates a charge-reduced TALEN variant that exhibits enhanced specificity. This parameter optimization reveals that reduced non-specific binding correlates with improved ability to distinguish target from off-target sequences.
3Reliability
If cationic amino acids are present in TALEN domains, then they facilitate DNA binding, but they also increase non-specific binding to off-target sequences
Solution Approach 1:
The patent applies local quality modification by specifically targeting the N-terminal and C-terminal domains for charge reduction while preserving the central TALE repeat array that contains the sequence-specific recognition residues. This localized modification strategy maintains specific DNA binding capability in the repeat region while reducing non-specific electrostatic interactions in the terminal domains, thereby improving overall specificity without compromising target recognition.
Solution Approach 2:
The invention changes the charge parameter of amino acid residues in specific domains from positive (cationic) to neutral or negative. By replacing lysine and arginine with glutamine, glycine, or other uncharged/negatively charged residues in the N-terminal and C-terminal domains, the patent reduces the net positive charge that mediates non-specific DNA binding, while preserving specific binding through the RVD-containing repeat array.
Data Source
AI summary
Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with improved specificity and methods for generating and using such TALEs.