Type V CRISPR Cas Proteins for Nucleic Acid Detection
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Solution Overview
Problem
Current systems for nucleic acid modification and detection are not suitable for samples with low concentrations of nucleic acids in complex mixtures, often requiring intensive sample preparation and being limited by accuracy and effectiveness in challenging biological sample conditions.
Innovation Solution
The use of Type V CRISPR/Cas proteins, coupled with guide nucleic acids, that provide cis cleavage, transcollateral cleavage, and nickase activities for specific modification and detection of nucleic acids across a wide range of sample conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional nucleic acid modification and detection systems are used, then they can perform basic functions, but they require intensive sample preparation and are not suitable for low concentration samples in complex mixtures
Solution Approach 1:
The patent employs Type V CRISPR/Cas proteins which fundamentally change the operational parameters of nucleic acid detection systems. These proteins can directly process low concentration samples without requiring extensive purification or concentration steps, thereby improving ease of operation while maintaining detection accuracy through their inherent specificity and catalytic activity.
2Reliability
If conventional detection systems are used, then they can detect nucleic acids, but they lack accuracy and effectiveness in challenging biological sample conditions
Solution Approach 1:
Type V CRISPR/Cas proteins exhibit multi-functionality by providing both cis cleavage activity (cleaving the target nucleic acid itself) and trans cleavage activity (cleaving reporter or indicator nucleic acids). This dual functionality allows the system to maintain high detection effectiveness across diverse sample conditions including high viscosity and metal chelating environments, thereby improving both reliability and adaptability.
3Measurement precision
If programmable nucleases are used, then they can provide sequence-specific cleavage, but their accuracy is limited by challenging biological sample conditions such as high viscosity and metal chelating
Solution Approach 1:
The patent utilizes trans cleavage activity where the Type V CRISPR/Cas protein cleaves a separate reporter or indicator nucleic acid instead of directly cleaving the target. This intermediary mechanism protects the target-specific binding accuracy from interference by challenging sample conditions, as the reporter cleavage occurs in a more controlled environment while still being triggered by specific target recognition.
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 rapid and accurate nucleic acid modification and detection with improved specificity and efficiency in diverse sample conditions, overcoming limitations of existing technologies.
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
A programmable nuclease may bind a target region of a nucleic acid and cleave the nucleic acid within the target region or at a position adjacent to the target region
Implementation Method 3
Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide RNA (crRNA or sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guideRNA
Implementation Method 4
Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide RNA. Trans cleavage activity is triggered by the hybridization of guide RNA to the target nucleic acid
Implementation Method 5
Nickase activity is the selective cleavage of one strand of a dsDNA molecule
Data Source
AI summary
The present disclosure provides compositions and methods of use for Type V CRISPR/Cas proteins. Type V CRISPR/Cas proteins may be configured to bind and modify nucleic acids in a sequence specific manner.