Temperature-Sensitive Mad7 Endonuclease for Reversible Gene Control
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Solution Overview
Problem
Current CRISPR genome editing systems lack precise control over gene expression regulation, particularly in temperature-dependent contexts, as existing RNA-guided endonucleases do not offer mechanisms to dynamically modulate their activity based on temperature changes.
Innovation Solution
Development of temperature-sensitive variants of the RNA-guided endonuclease Mad7, which undergo specific amino acid alterations to create temperature-sensitive complexes with guide-RNA and DNA, allowing for controlled binding and activity modulation by shifting temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If temperature-sensitive variants of Mad7 are introduced to enable dynamic control of gene expression, then adaptability is improved, but protein stability deteriorates at restrictive temperatures
Solution Approach 1:
The patent applies the dynamics principle by creating temperature-sensitive variants of Mad7 that can dynamically change their functional state in response to temperature changes. The tsMad7 variants transition from a stable, active state at permissive temperatures to an unstable, inactive state at restrictive temperatures, enabling reversible control of gene expression without permanent modification to the protein structure.
Solution Approach 2:
The patent employs parameter changes by introducing specific amino acid substitutions (e.g., D272N, D272K, D272R) that alter the thermal stability parameters of Mad7. These point mutations change the protein's conformational stability threshold, allowing it to maintain structure and function below a critical temperature while becoming unstable and inactive above that temperature, thus achieving temperature-dependent control.
2Ease of operation
If amino acid alterations are introduced to create temperature-sensitive complexes, then ease of operation is improved, but manufacturing precision deteriorates
Solution Approach 1:
The patent applies local quality by introducing targeted amino acid substitutions at specific positions within the Mad7 protein sequence (e.g., position 272). Rather than globally altering the protein, precise point mutations are made at critical residues that govern thermal stability, maintaining overall protein integrity while conferring temperature-sensitive behavior at specific functional sites.
3Reliability
If temperature-sensitive Mad7 variants are used for reversible gene regulation, then reliability is improved, but loss of time increases due to temperature cycling
Solution Approach 1:
The patent applies preliminary action by pre-engineering the temperature-sensitive properties into the Mad7 variant during protein construction. The tsMad7 variants are designed in advance with specific amino acid substitutions that predetermined their thermal response characteristics, allowing them to automatically and rapidly respond to temperature changes without requiring additional activation steps or prolonged equilibration periods.
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
Enables precise temporal and spatial control of gene expression by forming stable complexes at permissive temperatures and dissociating at restrictive temperatures, facilitating reversible regulation of target DNA sequences.
Implementation Method 1
introduction of specific amino acid alterations in the RNA-guided endonuclease Mad7 results in temperature-sensitive Mad7 variants (tsMad7). When employing these temperature-sensitive variants in vivo together with a suitable gRNA and a target DNA sequence, the initial variant-gRNA complex as well as the variant-gRNA-DNA complex become temperature-sensitive.
Data Source
AI summary
The present invention relates to temperature-sensitive variants of an RNA-guided endonuclease, polynucleotides encoding said variants, nucleic acid constructs and expression vectors comprising polynucleotides encoding said variants, host cells expressing said variant, methods of transient repression and expression of one or more DNA target sequence using said variants, and use of said variants, polynucleotides, nucleic acid constructs, expression vectors, host cells, and methods.