Modular Nucleic Acid Binding Domains for Precise Genome Editing
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Solution Overview
Problem
Existing genome editing and gene regulation techniques lack the precision and specificity required to effectively target and modify specific nucleic acid sequences, leading to off-target effects and inefficiencies.
Innovation Solution
Development of modular nucleic acid binding domains derived from bacteria such as Ralstonia and Xanthomonas, which comprise repeat units that can be engineered to bind specifically to target nucleic acid sequences with high affinity and specificity, allowing for precise genome editing and gene regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing genome editing techniques are used, then genome editing can be performed, but precision and specificity are insufficient leading to off-target effects
Solution Approach 1:
The nucleic acid binding domain is divided into multiple repeat units, where each repeat unit independently recognizes and binds to a specific nucleotide base. This segmentation allows for precise targeting of the desired sequence while avoiding off-target effects, as each repeat unit contributes to the overall specificity of the binding domain.
Solution Approach 2:
Different repeat units within the binding domain are designed with specific amino acid sequences that confer local binding preferences for different nucleotide bases (e.g., NG for guanine, HD for cytosine). This local quality variation across repeat units enables the binding domain to achieve high specificity for the target sequence by combining multiple localized recognition events.
2Manufacturing precision
If existing genome editing techniques are used, then genome editing can be performed, but specificity for target site is insufficient
Solution Approach 1:
The binding domain is segmented into multiple repeat units that work together to achieve high specificity. Each repeat unit targets a specific nucleotide, and the combination of multiple such units creates a highly specific recognition pattern that minimizes off-target binding while maintaining reliability.
Solution Approach 2:
The binding domain is constructed as a composite of multiple repeat units with different nucleotide recognition specificities. This composite structure allows the binding domain to achieve high target site specificity by combining the recognition capabilities of individual repeat units, thereby reducing off-target effects and improving overall reliability.
3Manufacturing precision
If modular nucleic acid binding domains with high potency are used, then precise targeting is achieved, but the complexity of the polypeptide structure increases
Solution Approach 1:
The binding domain is segmented into standardized repeat units of approximately 34 amino acids each. This segmentation into modular, repeating elements simplifies the design process despite the overall complexity, as each unit follows a consistent structure with variable regions that determine nucleotide specificity. The repetitive nature of the units provides a degree of structural predictability.
Solution Approach 2:
The repeat units are designed with a universal backbone structure that performs common functions (binding to the DNA major groove, maintaining structural stability), while only specific residues vary to provide nucleotide recognition. This universality reduces the overall complexity by separating the invariant structural framework from the variable recognition elements.
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
The modular nucleic acid binding domains achieve high potency and specificity, enabling precise targeting and modification of nucleic acid sequences with reduced off-target effects, enhancing the efficacy of genome editing and gene regulation processes.
Implementation Method 1
at least one repeat unit of the plurality of repeat units comprises a binding region configured to bind to a target nucleic acid base within the target site
Data Source
AI summary
Provided herein are polypeptides, compositions comprising the polypeptides and methods for genome editing and gene regulation (e.g., activation and/or repression) using the polypeptides or the compositions comprising the polypeptides, such as, DNA binding domains derived from the genus of Ralstonia. Also disclosed are DNA binding proteins that include a fragment of N-cap sequence of a TALE protein, such as, a Xanthomonas TALE protein. Also disclosed are DNA binding proteins that include a fragment of N-cap sequence of a DNA binding protein derived from bacteria of the genus Ralstonia.