Inducible Split Recombinases for Low-Toxicity Gene Expression Control
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Solution Overview
Problem
The toxicity of recombinases limits their use in manipulating gene expression in engineered cells, particularly for applications like AAV production systems.
Innovation Solution
Development of split recombinases with inducible recombinase activity, comprising a first and second polypeptide that lack activity independently but form a functional dimer upon dimerization, often mediated by a small molecule inducer, allowing controlled gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If recombinases are used to manipulate gene expression in engineered cells, then gene expression control is achieved, but cell toxicity increases
Solution Approach 1:
The recombinase enzyme is divided into two separate polypeptides (first and second polypeptides) that each lack recombinase activity on their own. These segmented polypeptides can be independently expressed in the cell and only form the active recombinase complex when both are present, thereby reducing toxicity while maintaining gene expression control capability.
2Object-affected harmful factors
If split recombinases with inducible activity are used, then toxicity is reduced, but system complexity increases
Solution Approach 1:
The first and second polypeptides are designed to dimerize and form a functional recombinase complex only when induced by a small molecule. This merging of two inactive polypeptides into an active complex upon induction allows reduced toxicity (since the complex forms only when needed) while the induction mechanism provides controlled activation.
Solution Approach 2:
The recombinase activity is made inducible through small molecule binding, which changes the conformational state of the polypeptide dimer from inactive to active. This parameter change (binding of inducer molecule) allows the system to transition between toxic and safe states, reducing overall toxicity while maintaining the ability to activate gene expression when required.
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
The split recombinases provide reduced toxicity and enable precise regulation of gene expression, facilitating the use of cytostatic or cytotoxic AAV gene products in AAV production systems.
Implementation Method 1
a recombinase dimer comprising the first polypeptide and the second polypeptide has recombinase activity
Data Source
AI summary
Described herein are split recombinases having inducible recombinase activity and polynucleic acid molecules encoding the same. Also described herein are kits and host cells comprising the split recombinases, as well as methods of their use.