Capturing ctDNA from Unaltered Stationary and Flowing Plasma with dCas9

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Liquid biopsies offer a non-invasive method for early cancer detection by analyzing circulating tumor DNA (ctDNA).
• Current ctDNA detection methods are limited by the small blood volume required and the need to alter plasma for DNA capture.
• Apheresis technology allows screening of large plasma volumes in vivo, but requires compatible ctDNA capture techniques.

Purpose of the Study:

• To develop the first technology capable of capturing ctDNA from flowing, unaltered plasma.
• To overcome the limitations of current ctDNA detection methods for early cancer diagnosis.

Main Methods:

• Utilized catalytically dead Cas9 (dCas9) and guide RNA for allele-specific capture of BRAF T1799A (BRAFMut) DNA.
• Simulated cancer patient plasma by spiking BRAFMut DNA into healthy donor plasma.
• Evaluated dCas9 capture efficiency in stationary and flowing (laminar and turbulent) unaltered plasma at different temperatures.
• Compared dCas9 performance against a commercial cell-free DNA (cfDNA) capture kit.

Main Results:

• dCas9 demonstrated efficient BRAFMut allele capture at both room temperature (25 °C) and body temperature (37 °C).
• In stationary plasma, dCas9 showed comparable efficiency to a commercial cfDNA kit but achieved 1.8-3.3-fold enrichment of BRAFMut.
• Turbulent flow significantly increased the ctDNA capture rate compared to laminar flow and stationary plasma, showing a linear relationship with time (R² = 0.874).

Conclusions:

• The developed dCas9 system can capture ctDNA from unaltered, flowing plasma.
• This technology holds potential for advancing early cancer detection through large-volume plasma screening.
• Further optimization and clinical validation are necessary to determine the full utility of this dCas9-based ctDNA capture system.