TALE Repeat Assembly via Type IIS Enzymes and Magnetic Beads
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Solution Overview
Problem
Current methods for assembling long arrays of TALE repeat domains are challenging due to the highly repetitive nature of these constructs, which can be unstable and prone to recombination, and require complex hierarchical assembly processes that are difficult to automate.
Innovation Solution
The use of Type IIS restriction enzymes and solid supports like magnetic streptavidin-coated beads for assembling TALE repeat domains, allowing for the creation of long arrays through sequential ligation and release steps, with restriction sites for efficient manipulation and automation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If hierarchical assembly processes are used to assemble long arrays of TALE repeat domains, then the assembly can be performed step-by-step, but the process becomes complex and difficult to automate
Solution Approach 1:
The TALE repeat array assembly is divided into modular repeat units, each flanked by unique Type IIS restriction sites. This segmentation allows standardized, automated assembly through sequential ligation of predefined modules, reducing overall process complexity while maintaining ease of manufacture.
Solution Approach 2:
Type IIS restriction enzymes serve as intermediaries that recognize specific sequences outside the insert region, enabling precise cutting and ligation of TALE repeat modules. This intermediary mechanism standardizes the assembly process, making it both simple to perform and amenable to automation.
2Reliability
If traditional cloning approaches are used for assembling TALE repeats, then the method is well-established, but the repetitive constructs become unstable and prone to recombination
Solution Approach 1:
Unique restriction sites are placed locally at the boundaries of each TALE repeat module, while the internal repetitive sequences remain unchanged. This local differentiation prevents recombination between repeats during cloning, enhancing construct stability without complicating the overall assembly approach.
Solution Approach 2:
The restriction site sequences are specifically designed to be recognized by Type IIS enzymes, changing the cutting parameter from within-gene to outside-gene locations. This parameter change stabilizes the repetitive constructs by preventing slippage and recombination while maintaining ease of assembly through standard cloning techniques.
3Manufacturing precision
If Type IIS restriction enzymes are used for assembly, then precise cutting outside insert regions is achieved, but additional enzymatic steps are required
Solution Approach 1:
Type IIS restriction sites are pre-designed and incorporated into the TALE repeat module sequences during plasmid construction. This preliminary action ensures that subsequent enzymatic cutting occurs at precise, predetermined locations outside the insert, achieving high manufacturing precision without requiring complex real-time adjustments.
Solution Approach 2:
Multiple TALE repeat modules are designed with compatible Type IIS restriction sites that can be assembled in a standardized sequence. This universality allows the same enzymatic system to handle diverse repeat combinations, reducing overall process complexity despite the added precision steps.
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 the stable assembly of long TALE repeat arrays with reduced risk of unwanted mutations, facilitating the generation of custom TALE proteins for specific DNA binding applications, such as targeted genome editing and gene regulation.
Implementation Method 1
provision of a first nucleic acid encoding one or more TALE repeat domains, having a 5' overhang created by a type IIS restriction enzyme
Implementation Method 2
solid supports like magnetic streptavidin-coated beads
Implementation Method 3
magnetic streptavidin-coated beads
Implementation Method 4
sequential ligation and release steps
Data Source
Figure 1~2
Figure 3
Figure 4A~4B
AI summary
The disclosure describes methods that include providing a first nucleic acid having a sequence encoding a first set comprising one or more transcription activator-like effector (TALE) repeat domains and/or one or more portions of one or more TALE repeat domains; contacting the first nucleic acid with a first enzyme, wherein the first enzyme creates a first ligatable end; providing a second nucleic acid having a sequence encoding a second set comprising one or more TALE repeat domains and/or one or more portions of one or more TALE repeat domains; contacting the second nucleic acid with a second enzyme, wherein the second enzyme creates a second ligatable end, and wherein the first and second ligatable ends are compatible; and ligating the first and second nucleic acids through the first and second ligatable ends to produce a first ligated nucleic acid, wherein the first ligated nucleic acid is linked to a solid support, and wherein the first ligated nucleic acid encodes a polypeptide comprising said first and second sets.