Researchers at Korea University College of Medicine have developed a liquid biopsy method that can detect cancer mutations present in very low frequencies from all cell-free DNA in a typical blood sample. The ultrasensitive method, called MUTE-Seq, uses FnCas9-AF2, an engineered high-fidelity CRISPR enzyme that depletes wild-type DNA in the sample before sequencing allowing for circulating tumor DNA (ctDNA) detection below 0.1% of total cfDNA, exceeding current detection limits. Details of this new approach are published in the journal Advanced Materials.
“Our findings suggest that the MUTE-Seq method has considerable potential for developing diagnosis panels aimed at detecting multiple low-frequency ctDNA for MCED, CDx, or MRD monitoring,” said senior author Junseok W. Hur, MD, PhD, a clinical assistant professor of medicine at Korean University.
Hur and his team embarked on their research in an attempt to improve current liquid biopsy approaches which often fail if the ctDNA in a blood sample is below 0.1% of total cell-free DNA. “During the early stages of cancer, ctDNA in the blood can be exceedingly minute (as low as 0.01% of total cfDNA), posing a significant challenge for detection,” the researchers wrote.
The team focused on a CRISPR-based enrichment method since they have been shown to deplete abundant non-mutated DNA sequences, thereby enhancing the visibility of rare mutations. But previous CRISPR-based enrichment methods including CARM, DASH, MAD-DASH, and CUT-PCR, exhibited limited utility since “the eliminations required the sequences to contain significant sequence differences, or the mutations to be positioned within the protospacer adjacent motif (PAM) site” and because previous CRISPR systems could not reliably distinguish single-base mismatches, the team noted.
To overcome this, the investigators “engineered a highly precise advanced-fidelity FnCas9 variant, named FnCas9-AF2, to effectively discriminate single-base mismatches at all positions of the single guide RNA (sgRNA) target sequences,” they wrote.
MUTE-Seq uses FnCas9-AF2 to cleave perfectly matched wild-type DNA before sequencing, thereby enriching the relative amount of mutant alleles. The depletion step prior to sequencing allows low-frequency variants to appear at levels that fall within confidence limits of standard sequencing, reducing the need for ultra-deep sequencing or extensive UMI barcoding.
To test their method, the researchers employed Sanger sequencing and next-generation sequencing and showed that MUTE-Seq increased variant allele frequencies by up to ten fold while providing a detection limit of around 0.005%. Testing this method in patients with acute myeloid leukemia, MUTE-Seq enabled minimal residual disease (MRD) assessment by identifying NRAS mutations that are typically undetectable using current techniques. And in multiplexed experiments using cfDNA from non-small cell lung cancer and pancreatic cancer patients, MUTE-Seq increased concordance between plasma and tumor tissue. In short, the new method “demonstrated a significant improvement in the sensitivity of simultaneous mutant detection and highlighted its clinical utility for early-stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA),” the investigators noted.
Next steps to further develop MUTE-Seq will focus on expanding hotspot panels for early multi-cancer detection, leveraging the technology to improve tumor-informed MRD monitoring, and developing protocols to integrate MUTE-Seq within existing sequencing workflows.
