TAL Effector Assembly Platform for Precise Genome Engineering
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Solution Overview
Problem
Current genome engineering tools, such as ZFNs and meganucleases, face challenges with off-target cleavage, limited specificity, and difficulty in predicting and designing binders, which hinders their flexibility and reliability for genetic manipulation.
Innovation Solution
The development of compositions and methods for designing and assembling nucleic acid molecules, including TAL effector molecules, to efficiently bind to specific DNA sequences, using linear nucleic acid molecules with covalently bound topoisomerase and recombination sites, allowing for precise genetic manipulation and modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If ZFNs and meganucleases are used for genome engineering, then genetic manipulation capability is achieved, but off-target cleavage and limited specificity occur
Solution Approach 1:
The TAL effector is divided into multiple repeat units, each recognizing a specific DNA base pair. This segmentation allows precise customization of DNA binding specificity by arranging different repeat types in a defined sequence, thereby achieving high target specificity while minimizing off-target effects
Solution Approach 2:
Each TAL repeat unit has specific amino acid residues at positions 12 and 13 (RVD) that determine base recognition specificity. By locally optimizing these residues in each repeat, the system achieves high specificity for the intended target sequence while maintaining overall protein stability and function
2Adaptability or versatility
If traditional genome engineering tools are used, then DNA binding capability is achieved, but difficulty in predicting and designing binders occurs
Solution Approach 1:
The invention establishes a direct code between amino acid sequences in TAL repeats and the DNA bases they recognize. By changing the RVD parameters in each repeat, researchers can predictably design binders for any desired DNA sequence, greatly simplifying the design process while maintaining adaptability to different target sequences
3Manufacturing precision
If customized TAL effector molecules are assembled, then precision of genetic engineering is improved, but assembly complexity increases
Solution Approach 1:
The TAL effector coding sequence is divided into multiple repeat-coding modules that can be independently synthesized and assembled. This modular segmentation enables precise assembly of customized effectors targeting specific DNA sequences while managing the complexity through standardized module interfaces
Solution Approach 2:
The repeat units are pre-designed and synthesized with correct sequences before assembly. This preliminary preparation of standardized modules simplifies the final assembly process and ensures precision in the resulting customized TAL effector molecule
Data Source
AI summary
The invention generally relates to compositions and methods for designing and producing functional DNA binding effector molecules and associated customized services, tool kits and functional assays. In some aspects, the invention provides methods and tools for efficient assembly of customized TAL effector molecules. Furthermore, the invention relates to uses of TAL effector molecules and functional evaluation of such TAL by, for example, customized assays.