A study from the Korea University College of Medicine, published in Advanced Materials shows that a novel CRISPR-based method significantly improves liquid biopsy sensitivity, detecting rare cancer DNA at very low levels.

Key Points

·       Higher sensitivity: MUTE-Seq detects cancer DNA at ultra-low levels.

·       CRISPR precision: FnCas9-AF2 enriches tumour DNA and reduces sequencing noise.

·       Clinical promise: Enables early detection and better treatment monitoring.

Role of FnCas9-AF2 and Developing MUTE-Seq

Although liquid biopsies are showing increasing promise for cancer detection and treatment monitoring, their effectiveness is typically limited by extremely low levels of tumour-derived DNA circulating in blood.

As a potential solution, researchers led by Professor Junseok W Hur from Korea University College of Medicine, in collaboration with multiple partners, developed MUTE-Seq. MUTE-seq is classed as a highly sensitive CRISPR-based method designed to detect cancer mutations present at exceptionally low frequencies while simultaneously reducing sequencing costs and background error noise.

Their approach relied on FnCas9-AF2, an engineered CRISPR enzyme built to recognise and discriminate even single-base mismatches with remarkable precision. Additionally, the variant shows near-zero off-target activity and selectively cleaves perfectly matched wild-type DNA, thereby enriching the relative proportion of circulating tumour DNA before sequencing. The enrichment allows rare variants to rise above the noise that often hinders next-generation sequencing.

Professor Hur stated: “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.”

Results

Sanger sequencing and next-generation sequencing were used to evaluate the performance of MUTE-Seq, which showed increased variant allele frequencies by up to ten times. This led to the detection of mutations present at approximately 0.005%, a level typically obscured by baseline sequencing error rates.

In patients with acute myeloid leukaemia, the method successfully identified minimal residual disease by amplifying weak NRAS mutation signals that are usually undetectable. When applied in multiplex mode across multiple clinically relevant cancer hotspots as EGFR and KRAS, MUTE-Seq improved concordance between plasma and tumour tissue in patients with non-small cell lung cancer and pancreatic cancer, including early-stage cases in which ctDNA levels are extremely low.

Conclusion

Overall, these findings showcase the broad potential of MUTE-seq to bolster the accuracy of liquid biopsy testing. MUTE-Seq offers a more reliable path for multi-cancer early detection (MCED) and tracking of treatment-emergent resistance mutations. Together, these advantages position MUTE-Seq as a scalable and clinically adaptable tool with significant promise for improving precision oncology.