Engineered TALE Polypeptides for Precise Genome Targeting
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Solution Overview
Problem
Current methods for site-specific genome manipulation in mammals 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, specifically ordered to target genomic loci, which include N-terminal and C-terminal capping regions and functional protein domains such as repressors, activators, and nucleases, to facilitate site-specific gene editing and regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If current methods for site-specific genome manipulation are used, then genome editing can be performed, but efficiency and precision are insufficient
Solution Approach 1:
The TALE protein is divided into multiple repeating monomers, each recognizing a specific nucleotide sequence. This segmentation allows the protein to target specific genomic locations with high precision while maintaining efficient binding through the cumulative effect of multiple recognition units.
Solution Approach 2:
The invention combines TALE monomers with functional domains (such as FokI nuclease) to create composite proteins that兼具 sequence-specific binding capability and genome editing function, achieving both high precision targeting and efficient genome manipulation.
2Measurement precision
If TALE monomers are specifically ordered to target genomic loci, then binding specificity increases, but protein structure complexity increases
Solution Approach 1:
The polypeptide is segmented into standardized monomer units with consistent structural motifs. Each monomer contains a conserved backbone structure with variable RVD sequences, allowing high binding specificity through sequence variation while maintaining structural simplicity through repetition of the same basic unit.
Solution Approach 2:
The invention varies the RVD parameters within each monomer to change binding specificity for different nucleotides, while keeping the overall protein structure parameters (monomer length, repeat structure) constant. This allows precise control of binding specificity without increasing structural complexity.
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 gene expression by preferentially binding to specific DNA sequences, allowing for site-specific gene editing and regulation with high specificity and accuracy.
Implementation Method 1
engineered DNA-binding polypeptides comprising Transcription activator-like effector (TALE) monomers and half-monomers, specifically ordered to target genomic loci
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.