dTALE Polypeptides for Precise Genome Editing
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Solution Overview
Problem
Current methods for manipulating the genome of target cells lack efficiency and precision, making systematic interrogation and engineering of biological systems difficult and costly.
Innovation Solution
Development of customized polypeptide sequences, known as designer transcription activator-like effectors (dTALE polypeptides), which act as sequence-specific nucleic acid binding proteins, allowing for precise targeting and modulation of gene expression through engineered nucleic acid molecules and expression vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing genome manipulation methods are used, then genome manipulation can be performed, but efficiency and precision are insufficient
Solution Approach 1:
The TALE protein is divided into multiple repeat units, each recognizing a specific nucleotide base. This segmentation allows the protein to be customized for different DNA sequences by simply changing the repeat units, thereby improving both precision and efficiency of genome manipulation.
Solution Approach 2:
The TALE protein structure serves multiple functions: the repeat region provides sequence-specific DNA binding, while the N-terminal and C-terminal regions provide effector functions such as transcription activation. This multi-functionality enables precise and efficient genome manipulation through a single protein design.
2Manufacturing precision
If customized polypeptide sequences are developed, then precision of nucleic acid binding is improved, but complexity of construction increases
Solution Approach 1:
The polypeptide is constructed from standardized repeat units that can be assembled in a defined order. Each repeat unit encodes specificity for one nucleotide, allowing precise binding through simple combinatorial assembly rather than complex de novo design.
Solution Approach 2:
The repeat units are pre-designed and standardized before assembly. The nucleotide recognition specificity is predetermined in each repeat unit, so the final precise binding capability is achieved through pre-prepared modular components rather than complex real-time construction.
3Reliability
If repetitive polypeptide sequences are used, then binding specificity is improved, but difficulty of assembly increases
Solution Approach 1:
The repetitive polypeptide sequence is divided into discrete repeat units that can be independently synthesized and then assembled in a controlled manner. This segmentation maintains binding specificity while enabling systematic assembly through standardized interfaces.
Solution Approach 2:
Each repeat unit is pre-synthesized with its specific nucleotide recognition capability established before assembly. The preliminary preparation of standardized repeat units simplifies the final assembly process while preserving the binding specificity required for reliable genome manipulation.
Data Source
AI summary
Provided herein are compositions, kits and methods useful in the construction of designer transcription activator-like effector (dTALE) polypeptides.