CRISPR Nucleic Acid Sensor With Point-of-Use crRNA Generation
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Solution Overview
Problem
Existing molecular diagnostics, such as PCR and CRISPR-based methods, are costly, bulky, and require specialized equipment, limiting their use to laboratory settings, while isothermal amplification methods suffer from false positives and RNA-based reagents are unstable, making decentralized molecular diagnostics impractical.
Innovation Solution
A CRISPR-based nucleic acid sensing platform that generates crRNA at the point of use using isothermal amplification, eliminating the need for pre-packaged RNA inputs and enabling various reporter signals, including colorimetric, electrochemical, enzymatic, and fluorescent modes, suitable for both cell-free and cell-based applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional imaging techniques are used to detect molecular interactions, then device complexity is reduced, but measurement precision and sensitivity are insufficient to detect weak or transient interactions
Solution Approach 1:
The system segments the detection function into separate modules: a reference well and an experimental well, each containing specific beads or surfaces. This segmentation allows independent optimization of each detection channel and enables parallel measurement of multiple parameters, thereby improving measurement precision without proportionally increasing overall system complexity.
Solution Approach 2:
The patent introduces intermediary elements such as beads functionalized with ligands or capture molecules that mediate between the molecular interactions of interest and the detection system. These intermediaries amplify weak signals and enable detection of transient interactions by providing stable, detectable readouts while maintaining relatively simple instrumentation.
2Adaptability or versatility
If conventional imaging techniques are used, then ease of operation is maintained, but the ability to quantify multiple molecular parameters simultaneously is limited
Solution Approach 1:
The system employs universal detection components that can measure multiple molecular parameters simultaneously. For example, beads can be functionalized with different ligands to detect multiple analytes, and the imaging system captures multiple parameters (binding affinity, kinetics, concentration) in a single experiment, enabling versatile multiplexed measurements while maintaining relatively simple operation through automated data analysis.
3Reliability
If traditional binding assays are used, then device complexity is low, but reliability is compromised by inability to control for non-specific binding and experimental variability
Solution Approach 1:
The patent merges the control and experimental measurements into a single integrated system. By incorporating reference wells with known binding characteristics alongside experimental wells, the system simultaneously measures and corrects for non-specific binding, background signals, and experimental variability, thereby improving reliability while the automated analysis reduces the perceived complexity of assay design.
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 platform provides robust and flexible molecular diagnostics capable of low detection thresholds and multiplexing, suitable for decentralized settings, without the need for RNA storage and with improved specificity and sensitivity.
Implementation Method 1
the sensor core to detect changes in refractive index that occur when a target molecule binds to a ligand
Implementation Method 2
patterns of light and dark fringes known as an interference pattern
Implementation Method 3
A laser source produces a beam of coherent light
Data Source
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AI summary
Provided are signal-inducing CRISPR-sensitive nucleic acid, optionally DNA, sensors, for example comprising: a) a non-functional CRISPR-sensitive DNA reporter construct comprising a non-functional expression cassette with at least one CRISPR target site inserted, generated by removal or addition of nucleic acids or naturally present in the expression cassette, the non-functional expression cassette having a reporter construct upstream end upstream of the CRISPR target site and a reporter construct downstream end downstream of the CRISPR target site, and b) a function-restoring nucleic acid, the function-restoring nucleic acid comprising an upstream flanking end, a function restoring repair insert and a downstream flanking end, wherein the upstream flanking end interfaces with reporter construct upstream end and/or the downstream flanking end interfaces with the reporter construct downstream end and one or both of the flanking ends are capable of permitting insertion or ligation of the function restoring repair insert into/to the reporter construct when the CRISPR target site is actuated under sensing condition, thereby producing a functional DNA reporter construct and sensor signal. Also provided are cell free and cell based systems, kits, primer pairs and molecular barcodes and methods of use thereof.