journal article·2025-11Open access

MUTE‐Seq: An Ultrasensitive Method for Detecting Low‐Frequency Mutations in cfDNA With Engineered Advanced‐Fidelity FnCas9

Sunghyeok Ye, Jin‐Soo Kim, Myungshin Kim, Ki‐Yeon Kim, Yoon‐Ho Won, Taegun Park, Sungjae An, Haerin Jeong, Hee‐Joon Chung, In Seon Lee, Myoung‐Hee Kang, Chan Young Kang, Mi Young Kim, Jae Ho Chung, Jeong‐An Gim, Woochang Hwang, Yonggoo Kim, Song Cheol Kim, Sungho Lee, Junho K Hur, Junseok W Hur

Advanced Materials· Volume 37· Issue 47

Abstract

Abstract In this study, we present the development of the Mutation tagging by CRISPR‐based Ultra‐precise Targeted Elimination in Sequencing (MUTE‐Seq) method. We 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. FnCas9‐AF2 exhibited significantly lower off‐target effects compared to existing high‐fidelity CRISPR‐Cas9 variants. MUTE‐Seq leverages FnCas9‐AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild‐type DNA, allowing for sensitive detection of low‐frequency cancer‐associated mutant alleles. MUTE‐Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE‐Seq was applied in a multiplexed manner on cell‐free DNA (cfDNA) from patients diagnosed with non‐small cell lung cancer (NSCLC) and pancreatic cancer. This approach 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). We anticipate that the FnCas9‐AF2‐based MUTE‐Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.
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Research context

OpenAlex topics: CRISPR and Genetic Engineering · Cancer Genomics and Diagnostics · Genomics and Phylogenetic Studies

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

Affiliations

GeneCker Seoul 04793 South Korea

Department of Internal Medicine Seoul National University Boramae Medical Center Seoul 07061 South Korea

Department of Laboratory Medicine College of Medicine The Catholic University of Korea Seoul 06591 South Korea

Catholic Genetic Laboratory Center Seoul St. Mary's Hospital College of Medicine The Catholic University of Korea Seoul 06591 South Korea

Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea

Department of Integrated Biomedical and Life Sciences College of Health Science Korea University Seoul 02841 South Korea

Center for Genomic Research and Development Korea University Anam Hospital Seoul 02841 South Korea

Department of Thoracic & Cardiovascular surgery College of Medicine Korea University Seoul 02841 South Korea

Department of Medical Science Soonchunhyang University Asan‐si Chungcheongnam‐do 31538 South Korea

Department of Pre‐Medicine College of Medicine Hanyang University Seoul 04763 South Korea

Hanyang Institute of Bioscience and Biotechnology Hanyang University Seoul 04763 South Korea

Division of Hepato‐Biliary and Pancreatic Surgery Department of Surgery BK21 Project Asan Medical Center University of Ulsan College of Medicine Seoul 05505 South Korea

Department of Genetics College of Medicine Hanyang University Seoul 04763 South Korea

Graduate School of Biomedical Science and Engineering Hanyang University Seoul 04763 South Korea

Funding

National Institute for International Education · Korea National Institute of Health · Korea Medical Device Development Fund · National Research Foundation of Korea · Korea University

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