TAL Effector Chimeric Nucleases for Precise DNA Targeting
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Solution Overview
Problem
Efficient methods for gene targeting in plants and animals have been difficult to achieve, limiting the ability to modify chromosomes through homologous recombination for crop improvement and disease treatment.
Innovation Solution
The use of transcription activator-like (TAL) effectors, which bind specifically to DNA sequences due to their repeat variable-diresidue (RVD) mechanism, enables the creation of chimeric nucleases that facilitate homologous recombination by introducing targeted double-stranded cuts in DNA, allowing for precise genetic modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional gene targeting methods are used, then chromosome modification can be achieved, but the efficiency is low and the process is difficult to accomplish
Solution Approach 1:
The patent combines TAL effector DNA-binding domains with FokI endonuclease domains to create chimeric nucleases. This merging of specific DNA-binding capability with cutting functionality enables efficient and targeted genome modification, resolving the contradiction between low efficiency and difficulty of traditional gene targeting methods.
Solution Approach 2:
The TAL effector acts as an intermediary that bridges specific DNA sequence recognition and the FokI endonuclease cutting activity. The TAL effector domain binds to the target DNA sequence and recruits the FokI domain to perform the cut, enabling precise gene targeting without the difficulties of traditional methods.
2Manufacturing precision
If TAL effector-DNA modifying enzyme is used, then precise DNA modification can be achieved, but the complexity of the system increases
Solution Approach 1:
The TAL effector system is segmented into modular repeat units, where each repeat recognizes a specific DNA base pair. This segmentation allows for precise customization of target sequences by simply changing the repeat sequence, achieving high precision DNA modification while maintaining system simplicity through modularity.
Solution Approach 2:
The patent utilizes parameter changes in the TAL effector repeat sequences (specifically the RVD motifs at positions 12 and 13) to alter DNA binding specificity. By changing these parameters, different target sequences can be recognized, enabling precise DNA modification at user-defined locations without increasing overall system 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
This approach enables precise and efficient genetic modifications, enhancing traits in plants for biofuels and biorenewables, and providing therapeutic applications with high specificity, such as targeting pathogens.
Implementation Method 1
the RVDs of TAL effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence
Implementation Method 2
a DNA modifying enzyme domain that can modify double stranded DNA
Implementation Method 3
providing to the cell a nucleic acid comprising a sequence homologous to at least a portion of the target DNA sequence, such that homologous recombination occurs between the target DNA sequence and the nucleic acid
Data Source
Figure 1A~1D
Figure 2A~2B
Figure 3
AI summary
Materials and methods related to gene targeting (e.g., gene targeting with transcription activator-like effector nucleases; "TALENS") are provided.