Protease-Conditional Cre Recombinase for Tight Activity Control
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Solution Overview
Problem
The always-on nature of Cre recombinase limits its applicability in genetic circuits for sensing and recording cellular events due to chromosomal rearrangements and altered cell physiology, and existing strategies for controlling its activity are not tight enough to prevent leaky expression or impair functionality.
Innovation Solution
Development of protease-conditional recombinases (ProRec) with engineered Cre recombinase variants that remain in a non-functional pro-enzyme state, using Nostoc punctiforme DnaE split intein-mediated protein cyclization, which are activated by proteolytic cleavage to enable precise control over recombinase activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cre recombinase is expressed continuously at high levels, then recombination efficiency is improved, but chromosomal rearrangements and altered cell physiology occur
Solution Approach 1:
The patent applies dynamics by transitioning Cre recombinase from a static always-on state to a dynamic regulated state. The recombinase is designed to switch between inactive and active states in response to cellular signals, allowing high recombination efficiency only when needed while preventing harmful chromosomal rearrangements during continuous expression.
Solution Approach 2:
The patent changes the activity parameter of Cre recombinase from constitutively active to conditionally active. By modifying the recombinase protein to respond to cellular signals, its activity level is dynamically adjusted based on cellular conditions, thereby achieving high recombination efficiency when required while avoiding harmful effects of continuous expression.
2Reliability
If Cre recombinase activity is controlled by existing strategies (promoter regulation, fusion proteins), then leaky expression is reduced, but recombination functionality is impaired
Solution Approach 1:
The patent extracts the regulatory function from the Cre recombinase protein itself by using a separate, inducible promoter system. This allows independent optimization of both control tightness (through the promoter) and recombination functionality (through the Cre protein), resolving the trade-off between reliable control and high productivity.
Solution Approach 2:
The patent introduces an intermediary mechanism (inducible promoter or signaling pathway) between the gene expression and Cre recombinase activity. This intermediary layer enables precise control of when and where Cre is expressed, maintaining tight regulation while preserving full recombination functionality when activated.
3Adaptability or versatility
If Cre recombinase is used for sensing and recording cellular events, then genetic circuit applications are enabled, but always-on nature prevents precise examination of development and disease
Solution Approach 1:
The patent applies periodic action by designing Cre recombinase to be activated in controlled temporal waves rather than continuously. The recombinase can be induced at specific time points to examine particular developmental stages or disease states, enabling precise measurement of cellular events at defined moments while maintaining versatility for various genetic circuit applications.
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 ProRec system provides a tightly controlled and highly functional recombinase variant that can detect viral or cellular proteolytic activity, offering a robust and permanent output signal for genetic circuit applications, enabling precise gene manipulation and infection monitoring.
Implementation Method 1
using Nostoc punctiforme DnaE split intein-mediated protein cyclization
Implementation Method 2
activated by proteolytic cleavage
Implementation Method 3
It uses a topoisomerase I-like mechanism to catalyze DNA recombination events between two specific DNA recognition sites, named loxP sites
Data Source
AI summary
The present disclosure relates to enzymes, compositions and methods for performing conditional homologous recombination of a targeted DNA molecule or genome by using modified proteins comprising nucleic acid DNA binding proteins with protease-conditional recombinase (hereinafter “ProRec”) activity. Namely a new cre recombinase pro enzyme, compositions/kit and sensors comprising the cre recombinase pro enzyme of the present disclosure. Namely, a cre recombinase pro-enzyme, for identifying and quantifying proteolytic activity in a disease or an infection, comprising intein mediated circularization, a flip-excision cassette comprising an output reporter and/or effector protein; a linker sequence cleavable by a protease.