Capturing ctDNA from Unaltered Stationary and Flowing Plasma with dCas9

Bradley M. Downs, Saraswati Sukumar

Research output: Contribution to journalArticlepeer-review


Many studies have established that blood-based liquid biopsies can be used to detect cancer in its early stages. However, the limiting factor for early cancer detection is the volume of blood required to capture the small amount of circulating tumor DNA (ctDNA). An apheresis machine is a device that can draw whole blood, separate the blood components, and infuse the blood components back into the individual. This device provides the opportunity to screen large volumes of plasma without extracting it from the body. However, current DNA capture technologies require the plasma to be altered before the ctDNA can be captured. Our goal was to develop the first technology that can capture ctDNA from flowing unaltered plasma. To simulate cancer patient plasma, we spiked BRAF T1799A (BRAFMut) DNA into plasma from healthy individuals. We used catalytically dead Cas9 (dCas9), guide RNA, and allele-specific quantitative polymerase chain reaction (qPCR) to capture and measure the number of captured BRAFMutDNA copies. We found that dCas9 captured BRAFMutalleles with equal efficiency at room temperature (25 °C) and body temperature (37 °C). Next, we showed that, in stationary unaltered plasma, dCas9 was as efficient in capturing BRAFMutas a commercial cell-free DNA (cfDNA) capture kit. However, in contrast to the cfDNA capture kit, dCas9 enriched BRAFMutby 1.8-3.3-fold. We then characterized the dCas9 capture system in laminar and turbulent flowing plasma. We showed that the capture rate using turbulent flow was greater than that in laminar flow and stationary plasma. With turbulent flow, the number of captured BRAFMutcopies doubles with time (slope = -1.035 Ct) and is highly linear (R2= 0.874). While we showed that the dCas9 capture system can capture ctDNA from unaltered flowing plasma, further optimization and validation of this technology is required before its clinical utility can be determined.

Original languageEnglish (US)
Pages (from-to)24113-24121
Number of pages9
JournalACS Applied Materials and Interfaces
Issue number21
StatePublished - Jun 1 2022


  • DNA capture
  • ctDNA
  • dCas9
  • liquid biopsy
  • magnetic beads
  • qPCR

ASJC Scopus subject areas

  • Materials Science(all)


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