Tunable Repeat Units for Genome Editing Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome editing and gene regulation techniques lack nucleic acid binding domains with strong and specific binding to target genes, limiting their efficacy and specificity.
Innovation Solution
Development of modular nucleic acid binding domains with tunable binding activity, comprising a plurality of repeat units that recognize target nucleic acid bases, and functional linker regions for dual activity compositions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current genome editing and gene regulation techniques are used, then basic genome editing function is achieved, but binding strength and specificity to target genes are insufficient
Solution Approach 1:
The nucleic acid binding domain is divided into multiple repeat units (e.g., 7-25 repeat units), where each repeat unit recognizes a specific target nucleic acid base. This segmentation allows for modular assembly of high-specificity binding domains that can be tailored to recognize specific target sequences with enhanced binding strength and specificity, resolving the contradiction between binding reliability and editing productivity.
Solution Approach 2:
The patent varies the number of repeat units (7-25 units) and their configurations to tune binding activity parameters. By changing the quantity and arrangement of repeat units, the binding strength and specificity are optimized to achieve both high reliability (specificity ratio of 50:1) and high productivity (indel percentage greater than 65%).
2Reliability
If modular nucleic acid binding domains with multiple repeat units are developed, then binding specificity is improved, but composition complexity increases
Solution Approach 1:
The complex binding domain is segmented into standardized repeat units that can be assembled in a modular fashion. Each repeat unit follows a consistent structure (A1-11X1X2B14-35 format) with specific functional regions, making the overall complex structure systematic rather than arbitrary. This modular segmentation reduces design complexity while maintaining high specificity.
Solution Approach 2:
The repeat units are designed with universal structural features and conserved regions (such as the B14-35 region) that can be interchangeably combined to create different binding specificities. This universality allows a single modular framework to achieve multiple binding targets, reducing the need for entirely different complex structures for each application.
3Adaptability or versatility
If repeat units are separated by linkers with recognition sites, then dual activities are achieved, but structural complexity increases
Solution Approach 1:
The linker regions are designed with universal recognition sites that can bind various molecules (small molecules, proteases, kinases) or serve as localization signals. This multi-functionality allows the same linker structure to provide diverse activities, reducing the need for different complex structures for different functions and actually simplifying the overall design.
Solution Approach 2:
The linkers act as intermediary elements that connect the nucleic acid binding domain to functional domains or provide additional regulatory activities. These intermediary regions with recognition sites mediate interactions with other molecules or domains, enabling dual activities without requiring direct integration of all functions into the binding domain itself, thus managing structural complexity.
Data Source
AI summary
Provided herein are DNA binding domains comprising a plurality of repeat units, wherein each repeat unit is expanded or contracted in length. Also provided herein are DNA binding domains comprising a plurality of repeat units, wherein each repeat unit is separated from a neighboring repeat unit by a linker. In certain aspects, the linker includes a recognition site. Also disclosed are DNA binding proteins that include a fragment of N-cap sequence of a TALE protein. The TALE protein may be a Xanthomonas TALE protein.