Cas9 Microfluidic ctDNA Capture from Low-Volume Plasma
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Solution Overview
Problem
The volume of plasma required to capture measurable levels of circulating tumor DNA (ctDNA) in blood-based liquid biopsies is a limiting factor for cancer detection and monitoring.
Innovation Solution
Devices and systems utilizing RNA-guided DNA binding proteins, such as Cas9, immobilized on microfluidic channels with mixing elements, capture ctDNA by hybridizing with guide RNAs, allowing for efficient capture and isolation of ctDNA from plasma samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional plasma processing methods are used to capture ctDNA, then DNA capture can be achieved, but large volumes of plasma are required which limits cancer detection efficiency
Solution Approach 1:
The patent replaces conventional mechanical/chemical DNA extraction methods with a CRISPR-based biological recognition system. The guide RNA specifically binds to ctDNA sequences through hybridization, enabling highly selective capture without requiring large plasma volumes. This biological recognition mechanism substitutes traditional mechanical separation and purification approaches.
Solution Approach 2:
The invention changes the binding affinity parameter by using engineered guide RNA with optimized complementarity to ctDNA targets. This increases the specificity and strength of DNA-capture complex formation, allowing efficient capture from smaller plasma volumes. The CRISPR system parameters (guide RNA sequence, Cas9 concentration) are optimized to maximize capture efficiency.
2Measurement precision
If CRISPR-based capture complexes are used, then selective ctDNA capture is improved, but device complexity increases due to immobilization requirements
Solution Approach 1:
The patent uses magnetic particles as intermediary carriers to hold the CRISPR capture complexes. These magnetic beads serve as a simple platform that can be easily manipulated with external magnets, avoiding complex immobilization structures. The magnetic intermediary simplifies the overall device design while maintaining high capture selectivity.
Solution Approach 2:
The CRISPR capture complexes are designed to be multi-functional: they provide specific DNA recognition through guide RNA, enable magnetic manipulation through coated particles, and allow flexible immobilization on various surfaces. This universality reduces device complexity by using a single versatile capture platform for multiple functions.
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 systems enable the selective capture of ctDNA from plasma without altering it, enabling its use for cancer detection and subsequent analysis, while allowing plasma to be returned to the subject for further cancer detection methods.
Implementation Method 1
a guide RNA configured to at least partially hybridize to a circulating free DNA (cfDNA) of interest
Implementation Method 2
an RNA-guided DNA binding protein, or a functional fragment thereof, bound to a guide RNA
Implementation Method 3
the plurality of capture complexes are magnetically immobilized to the one or more locations on the interior surfaces of the channel
Data Source
AI summary
The present disclosure provides devices, systems, kits, and related methods for isolating DNA from a sample (e.g., plasma) using RNA-guided DNA binding proteins (e.g., Cas proteins). Particularly, the disclosure provides devices, systems, kits, and related methods for Cas9 mediated capture of circulating free DNA (cfDNA) from flowing plasma.