Chemically Inducible Split-Recombinase Dimerization for Gene Circuits
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Solution Overview
Problem
Current methods for regulating gene expression in mammalian cells are limited by tissue or cell-specific promoters, leading to inefficiencies and challenges in designing complex genetic circuits with multiple inputs and outputs, and there is a lack of a generalizable platform for creating multi-input-multi-output genetic devices with minimal interconnecting layers and optimization.
Innovation Solution
The development of split-recombinases that are reconstituted by protein complementation in the presence of specific inducers, allowing for controlled gene expression independent of recombinase expression, using chemically-induced dimerization domains to bring together inactive fragments of recombinase proteins into functional recombinases for precise regulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If tissue or cell-specific promoters are used to regulate gene expression, then gene expression can be controlled in specific cell types, but the design of complex genetic circuits with multiple inputs and outputs becomes inefficient and challenging
Solution Approach 1:
The recombinase enzyme is divided into two separate inactive fragments (N-terminal and C-terminal portions). Each fragment is expressed from an independent inducible promoter, allowing controlled reconstitution of active recombinase only when both fragments are present. This segmentation enables complex logic circuit functionality while simplifying circuit design through modular, standardized components.
Solution Approach 2:
Chemically-induced dimerization domains (CIDDs) serve as intermediary elements that mediate the interaction between the two recombinase fragments. When induced by specific chemicals, the CIDDs facilitate dimerization and bring the inactive fragments together to form active recombinase, enabling precise control of gene expression without requiring complex promoter arrangements.
2Reliability
If traditional promoter-based regulation is used, then gene expression can be controlled, but extensive fine-tuning is required to engineer functional circuits
Solution Approach 1:
The recombinase is pre-split into two inactive fragments with defined boundaries (e.g., amino acids 1-200 and 201-400). These fragments are prepared in advance and expressed from standardized inducible promoters. The splitting is done preliminarily to create modular units that can be reliably reconstituted without requiring extensive fine-tuning of promoter strength or expression levels.
Solution Approach 2:
The system changes the control parameter from promoter strength fine-tuning to chemical induction timing and concentration. By using inducible promoters that respond to well-defined chemical signals (e.g., doxycycline, tamoxifen), the system achieves reliable gene expression control through parameter changes in the induction regime rather than through complex promoter engineering.
3Adaptability or versatility
If the number of inputs and outputs in genetic circuits increases, then the functionality and decision-making capabilities of cells improve, but the number of transcription units and effort required to balance input-output responses increases significantly
Solution Approach 1:
The split-recombinase system provides a universal platform that can be applied to create multiple different logic gates and circuit configurations using the same basic components. The same N-terminal and C-terminal fragments with CIDDs can be combined with different inducible promoters and target genes to achieve various input-output relationships, reducing the need for separate transcription units for each circuit element.
Solution Approach 2:
The system adds a temporal and chemical dimension to circuit control by using inducible promoters responsive to different chemical signals. This allows multiple inputs to be integrated through temporal sequencing of chemical inductions rather than requiring parallel transcriptional regulation pathways, effectively adding a time dimension to the logic operations.
4Ease of operation
If split-recombinases with chemically-induced dimerization domains are used, then controlled gene expression independent of recombinase expression is achieved, but the system requires additional components and induction mechanisms
Solution Approach 1:
The system merges the inducible promoter control mechanism with the recombinase activity control. The CIDDs are fused to the recombinase fragments, combining the chemical induction signal transduction with the enzymatic function in a single integrated system. This reduces the need for separate induction and execution components.
Solution Approach 2:
The split-recombinase system with CIDDs is self-regulating through chemical induction. When the inducing chemical is present, it automatically triggers dimerization and reconstitution of active recombinase, which then performs the DNA recombination function. The system serves itself by using the chemical signal to directly control its own activation without requiring external regulatory proteins or complex control mechanisms.
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 engineering of complex logic circuits in mammalian cells with minimal optimization, accommodating multiple inputs and outputs without increasing design complexity, and provides a generalizable framework for sophisticated control over gene expression, expanding the phenotypic space and enhancing the robustness and scalability of genetic circuit designs.
Implementation Method 1
a first recombinase polypeptide fragment and a second recombinase polypeptide fragment, wherein the first and second recombinase polypeptide fragments are not active by themselves but rapidly complement to reconstitute the active recombinase protein
Implementation Method 2
each recombinase polypeptide fragment is conjugated to a chemically-induced dimerization domain
Data Source
AI summary
The technology described herein relates to controlled chemically- or light-induced rejoinder of split-recombinases. In some embodiments, compositions, methods, kits and systems are provided that relate to a split-recombinase system, whereby protein complementation or rejoinder of split-recombinases is mediated by chemical-induced dimerization domains (CIDDs) or light-induced dimerization domains (LIDD) and rejoinder of the split-recombinases occur in the presence of one or more chemical inducers or light inducers, respectively. The split-recombinases systems as disclosed herein can be used in gene therapy, integrated logic and memory in living cells such as mammalian cells. The nucleic acid cassettes, switches, and systems described herein allow for controlled gene expression or gene regulation. The controlled chemically- or light-induced rejoinder of split-recombinases can be used in, for example, adopted T-cell therapy.