Effector Proteins for Nucleic Acid Detection in High Viscosity
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Solution Overview
Problem
Programmable nucleases face challenges in maintaining specificity and efficiency across a wide range of biological sample conditions, particularly in high viscosity and metal chelating environments, limiting their accuracy and effectiveness in nucleic acid detection and modification.
Innovation Solution
Compositions and systems comprising CRISPR/Cas proteins with engineered guide nucleic acids and effector proteins, optimized for high temperatures and specific buffer conditions, enhance the specificity and efficiency of nucleic acid cleavage, detection, and modification, including the use of CRISPR/Cas proteins with amino acid sequences identical to SEQ IDs 1-4 and crRNA sequences identical to SEQ IDs 19-21, in conjunction with trans-activating crRNA and single guide RNA, in various buffers and additives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If programmable nucleases are used in biological samples, then nucleic acid detection and modification can be performed, but specificity and efficiency deteriorate under high viscosity and metal chelating conditions
Solution Approach 1:
The patent applies parameter changes by optimizing buffer conditions including pH, ionic strength, and metal ion composition to counteract the harmful effects of high viscosity and metal chelating environments. The effector proteins are engineered to function optimally under specific buffer conditions that maintain their activity despite challenging sample conditions.
Solution Approach 2:
The patent introduces engineered buffer systems and additive compositions as intermediaries between the effector proteins and the challenging biological sample conditions. These intermediaries protect the proteins from viscosity and metal chelation effects while allowing the nuclease activity to proceed effectively.
2Measurement precision
If standard CRISPR/Cas proteins are used, then nucleic acid cleavage can occur, but accuracy and effectiveness are limited under diverse sample conditions
Solution Approach 1:
The patent creates effector proteins with enhanced universal performance by engineering them to maintain high accuracy across multiple different sample conditions. The proteins are designed to function effectively in various buffer compositions, temperatures, and sample types, making the system universally applicable rather than condition-specific.
Solution Approach 2:
The patent introduces dynamic adaptability through engineered effector proteins that can adjust their conformational states and binding affinities in response to different environmental conditions. This dynamic behavior allows the proteins to maintain optimal function whether the sample is high viscosity, contains metal chelators, or varies in other parameters.
3Temperature
If effector proteins are engineered for high temperature operation, then diagnostic applications are enabled, but protein stability may be compromised
Solution Approach 1:
The patent applies parameter changes by engineering effector proteins with modified amino acid sequences that increase thermal stability. Specific residues are optimized for high-temperature function while maintaining structural integrity, and buffer conditions are adjusted to support protein stability at elevated temperatures up to 65°C or higher.
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
These compositions and systems enable precise and efficient detection and modification of nucleic acids at elevated temperatures, improving accuracy and reliability across diverse sample conditions, making them suitable for diagnostic applications.
Implementation Method 1
guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid
Implementation Method 2
Programmable nucleases are proteins that bind and cleave nucleic acids in a sequence-specific manner
Data Source
AI summary
The present disclosure provides compositions of effector proteins. The compositions may comprise engineered guide nucleic acids. Also disclosed are the methods and systems for detecting and modifying target nucleic acids using the same. The compositions may provide nucleic acid modification and/or detection at relatively high temperatures.