Engineered TALE Polypeptide Nucleotide Recognition
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Solution Overview
Problem
Current methods for site-specific genome manipulation in mammalian cells lack efficiency and precision, necessitating improved compositions and methods for targeting specific genomic sites.
Innovation Solution
The use of engineered DNA-binding polypeptides comprising Transcription activator-like effector (TALE) monomers and half-monomers, arranged in a predetermined N-terminus to C-terminus orientation, to specifically target and manipulate genomic loci through mechanisms such as gene repression, activation, or site-specific editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for site-specific genome manipulation are used, then genome manipulation can be performed, but efficiency and precision are insufficient
Solution Approach 1:
The TALE protein is divided into multiple repeating monomer units, each recognizing a specific nucleotide sequence. This segmentation allows the protein to target specific genomic loci with high precision while maintaining efficient binding through the cumulative effect of multiple recognition modules working together in sequence
Solution Approach 2:
Each TALE monomer is engineered with specific local properties (amino acid sequences at positions 12 and 13) that determine its nucleotide recognition specificity. This local quality variation across different monomers enables the polypeptide to achieve both precise targeting of specific sequences and efficient binding through optimized local interactions
2Measurement precision
If TALE polypeptides are engineered to target specific nucleotide sequences, then binding specificity is improved, but the complexity of designing and constructing the polypeptides increases
Solution Approach 1:
The complex TALE polypeptide is constructed by assembling standardized monomer units, each with a defined nucleotide recognition code. This segmentation into modular components simplifies the design process, as researchers can select and combine pre-characterized monomers with known specificities rather than designing entire polypeptides from scratch
Solution Approach 2:
The TALE monomer structure serves multiple functions: it provides the structural framework for DNA binding, contains the nucleotide-specific recognition code in its variable regions, and can be systematically combined with other monomers to recognize different sequences. This universality reduces construction complexity by using a single modular design paradigm for all TALE polypeptides
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
Enables precise and efficient manipulation of genomic loci in mammalian cells, allowing for altered gene expression and editing by preferential binding and activation or repression of targeted genomic sequences.
Implementation Method 1
a DNA binding domain which may comprise at least five or more TALE monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest
Data Source
AI summary
The invention relates to methods of altering expression of a genomic locus of interest or specifically targeting a genomic locus of interest in an animal cell, which may involve contacting the genomic locus with a non-naturally occurring or engineered composition that includes a deoxyribonucleic acid (DNA) binding polypeptide having a N-terminal capping region, a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein the polypeptide includes at least one or more effector domains, and wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to the DNA of the genomic locus.