Intein-Assembled TALENs for Smaller Plasmids and Flexible Targeting
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Solution Overview
Problem
Existing genome editing technologies using rare-cutting nucleases like TALENs face challenges in efficient delivery and flexibility due to large plasmid sizes and inflexible target specificity, limiting their application in precise gene editing.
Innovation Solution
The use of inteins to splice and ligate separate half TALENs and rare-cutting nucleases, allowing for smaller molecular complexes and increased expression frequency, with flexible target specificity through complementary inteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If TALENs are delivered as complete fusion proteins, then target specificity is maintained, but plasmid size becomes large and delivery efficiency decreases
Solution Approach 1:
The TALEN system is divided into two separate components: a TALE protein and a nuclease domain. These are delivered as separate entities that can independently traverse the cell membrane and subsequently assemble through intein-mediated splicing to form the functional TALEN fusion protein, thereby reducing individual plasmid sizes while maintaining target specificity
Solution Approach 2:
An intein sequence is introduced as an intermediary element that facilitates the post-translational assembly of the TALE and nuclease components. The intein enables spontaneous splicing and ligation of the separate proteins after they enter the cell, allowing the formation of the complete TALEN fusion protein without requiring large plasmids to encode the entire fusion sequence
2Weight of stationary object
If TALEN components are delivered separately, then plasmid size is reduced and delivery efficiency improves, but assembly complexity increases
Solution Approach 1:
The intein sequences are designed to enable self-splicing and self-ligation of the TALE and nuclease components after they enter the cell. This autonomous post-translational assembly process eliminates the need for complex external assembly procedures, reducing assembly complexity while allowing separate delivery of smaller plasmid components
3Measurement precision
If traditional TALEN design is used, then target specificity is fixed, but flexibility in targeting different sequences is limited
Solution Approach 1:
The TALE component is designed with modular repeats that can be dynamically reconfigured to recognize different DNA sequences. The intein-mediated assembly system allows for flexible combination of different TALE variants with the nuclease domain, enabling adaptation to various target sequences while maintaining precise binding through the modular TALE structure
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 enhances the efficiency and flexibility of gene editing by reducing plasmid size and improving target specificity, facilitating more effective transformation and editing of genetic material in cells.
Implementation Method 1
the first intein and the second intein are configured to self-splice when in contact and, in response, to form a first half transcription activator-like effector nuclease (TALEN) including the first TALE bound to the rare-cutting nuclease
Data Source
AI summary
Embodiments of the present disclosure are directed to a plurality of nucleotide sequences encoding a first intein fused to at least a portion of a first transcription activator-like effector (TALE), a second nucleotide sequence encoding the first intein fused to at least a portion of a second TALE, and a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease.