Ligation-Based Cas Activation for Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid detection systems that utilize Cas collateral cleavage activity require specific guide RNA design for each target sequence, necessitating significant development burdens and introducing potential sequence mutations through amplification, which can lead to false results.
Innovation Solution
Decoupling target sequence detection from Cas cleavage activation using a ligation-based system that generates a Cas-activating nucleic acid, allowing a single guide RNA to detect multiple targets without specific hybridization, and eliminating the need for initial target amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification of target nucleic acid is performed to enhance detection sensitivity, then detection sensitivity is improved, but sequence mutation errors occur leading to false results
Solution Approach 1:
Instead of amplifying the target nucleic acid first and then detecting it, the invention inverts the approach by using a ligation-based system where the target nucleic acid directly activates Cas enzyme through specific hybridization and ligation events, eliminating the need for separate amplification steps and avoiding amplification-induced mutations
2Measurement precision
If specific guide RNA is designed for each target sequence to achieve specific detection, then detection specificity is improved, but device complexity and development burden increase
Solution Approach 1:
The invention creates a universal detection system where a single guide RNA can detect multiple different target sequences. The guide RNA is designed to hybridize with a conserved region or structure that is common across multiple targets, allowing one guide RNA to serve multiple detection functions through the ligation-based activation mechanism
Solution Approach 2:
The invention introduces a ligation-based intermediary system between target recognition and Cas activation. The ligation event acts as a mediator that converts diverse target sequences into a unified activation signal for the Cas enzyme, decoupling target specificity from guide RNA design
3Reliability
If ligation-based system is used for target detection without amplification, then sequence mutation errors are eliminated, but detection sensitivity may be reduced
Solution Approach 1:
The invention performs preliminary ligation and activation events before the actual Cas cleavage reaction. The ligation-based system prepares the activating nucleic acid in advance, ensuring that when Cas is activated, it is already in a state optimized for sensitive detection without requiring subsequent amplification steps
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 sensitive and specific detection of nucleic acids down to single base discrimination without amplification, reducing sequence mutation errors and simplifying guide RNA design, while allowing flexibility in using either DNA or RNA as activating nucleic acids.
Implementation Method 1
utilizes a ligation-based system for initial target nucleic acid hybridization, where components of this ligation-based system are designed so that the ligation event generates nucleic acid molecule(s) that is/are, or that template, activating nucleic acids for Cas collateral cleavage activity
Implementation Method 2
utilize collateral cleavage activity of certain Cas enzymes
Implementation Method 3
permit, among other things, multimerization of Cas activation sequences (i.e., those bound by a gRNA/crRNA)
Data Source
Figure 1A
Figure 1B
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AI summary
The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence).