Thermostable CRISPR-Cas Proteins for One-Pot Nucleic Acid Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Certain Cas enzymes are insufficiently stable at elevated temperatures, limiting their effectiveness in nucleic acid extension and amplification reactions, and existing thermostable variants do not adequately address this issue in one-pot assays.
Innovation Solution
Development of thermostable Cas12 and Cas13 homologs with amino acid sequences having 80-100% identity to SEQ ID Nos. 1-10, which maintain collateral cleavage activity above 60-65°C, enabling single-vessel nucleic acid detection and amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas enzymes are used in nucleic acid extension and amplification reactions at elevated temperatures, then nucleic acid detection and amplification can be performed, but the Cas enzymes lose stability and insufficient collateral cleavage activity is maintained
Solution Approach 1:
The patent modifies the amino acid sequence parameters of Cas enzymes to create thermostable variants. Specifically, it identifies and implements amino acid substitutions that increase thermal stability while preserving collateral cleavage activity, allowing the enzymes to function reliably at elevated temperatures required for nucleic acid amplification reactions
Solution Approach 2:
The patent creates copies of Cas enzyme sequences with modified amino acid compositions. By generating thermostable variants through sequence copying and modification, the invention produces enzymes that replicate the essential functions of native Cas enzymes while exhibiting enhanced thermal stability for use in one-pot amplification assays
2Device complexity
If one-pot nucleic acid amplification and detection assays are performed, then reaction simplicity and speed are improved, but Cas enzyme activity becomes insufficient due to elevated temperature exposure
Solution Approach 1:
The patent changes the thermal stability parameters of Cas enzymes through amino acid sequence modification. This enables the enzymes to withstand the elevated temperatures inherent in one-pot amplification assays without losing catalytic activity, thereby maintaining reliability while preserving the simplicity of the one-pot format
Solution Approach 2:
The thermostable Cas enzyme variants developed in this patent can perform multiple functions within a single reaction vessel: they serve as both the amplification catalyst and the detection agent. This multi-functionality allows the same enzyme to reliably execute both nucleic acid amplification and collateral cleavage-based detection in one-pot assays
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 thermostable Cas proteins ensure stable collateral cleavage activity at elevated temperatures, facilitating efficient nucleic acid detection and amplification in a single reaction vessel, enhancing the reliability of diagnostic and therapeutic applications.
Implementation Method 1
The present disclosure provides improved CRISPR-Cas proteins characterized by thermostable activity and/or Cas-protein collateral activity
Implementation Method 2
certain Cas enzymes may be insufficiently stable to maintain a sufficient level of activity (e.g., collateral activity) under such conditions... Cas enzymes whose activity (e.g., collateral cleavage activity) is insufficiently stable to maintain sufficient activity through any and all elevated-temperature step(s)
Data Source
AI summary
The present disclosure provides improved CRISPR-Cas proteins (e.g., improved thermostability).