Modular Pathogen-Derived Binding Domains for Precise Genome Targeting
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Solution Overview
Problem
Existing genome editing and gene regulation techniques face challenges in achieving ease of production, target specificity, and versatility with nucleic acid binding domains.
Innovation Solution
Development of non-naturally occurring modular nucleic acid binding domains derived from animal pathogen proteins, specifically from Legionellales and Francisella bacteria, which comprise repeat units that recognize specific bases in target nucleic acids, allowing for precise genome editing, gene regulation, and imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional nucleic acid binding domains (zinc-finger proteins, TALE proteins) are used for genome editing, then target specificity can be achieved, but ease of production and versatility are limited
Solution Approach 1:
The nucleic acid binding domain is segmented into multiple repeat units, where each repeat unit independently recognizes a specific base in the target nucleic acid. This modular segmentation allows for easier production through standardized assembly of repeat units while maintaining high target specificity through the cumulative recognition capability of multiple units.
Solution Approach 2:
The repeat unit-based nucleic acid binding domain is designed to be universal and versatile, capable of targeting different nucleic acid sequences by simply changing the combination of repeat units. This multi-functional design enables the same platform to be used for various genome editing applications across different target sites, improving both ease of production and versatility simultaneously.
2Adaptability or versatility
If conventional nucleic acid binding domains are used, then genome editing capability is provided, but versatility for different applications (imaging, regulation) is limited
Solution Approach 1:
The nucleic acid binding domain with repeat units is designed as a universal platform that can be combined with different functional domains to achieve multiple applications including genome editing, imaging, and gene regulation. The repeat unit structure itself maintains target specificity while the modular design enables versatility across different functional contexts.
Solution Approach 2:
By segmenting the binding domain into independent repeat units that each recognize specific bases, the system allows for precise targeting while enabling combinatorial flexibility. This segmentation permits the same repeat unit library to be used across different applications by varying the functional domains attached, thus achieving both target specificity and versatility.
3Manufacturing precision
If highly specific nucleic acid binding is achieved with conventional domains, then genome editing precision is improved, but device complexity increases
Solution Approach 1:
The binding domain is divided into standardized repeat units with consistent structural features and defined base recognition specificities. This segmentation into uniform modules simplifies the overall design and assembly process compared to conventional domains, while maintaining high genome editing precision through the cumulative recognition of multiple repeat units targeting specific nucleic acid sequences.
Data Source
AI summary
Provided herein are polypeptides, compositions, and methods for use thereof for genetic and epigenomic engineering, including, genome editing and gene regulation. These polypeptides and compositions include nucleic acid binding domains that bind to a target nucleic acid of interest. The nucleic acid binding domains include repeat units derived from repeat units identified in proteins from animal pathogens such as bacterium of the order Legionellales and the species Legionella and Francisella.