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Adv. Mater., Aug 21 2025, | https://doi.org/10.1002/adma.202505208 MUTE-Seq: An Ultrasensitive Method for Detecting Low-Frequency Mutations in cfDNA With Engineered Advanced-Fidelity FnCas9

Sunghyeok Ye ^{1 †}, Jin-Soo Kim^{2 †}, Myungshin Kim ^{3 4 †}, Ki-Yeon Kim ¹, Yoon-Ho Won ¹, Taegun Park ^{1 5 6}, Sungjae An ⁵, Haerin Jeong ¹, Hee-Joon Chung ¹, In Seon Lee ¹, Myoung-Hee Kang ^{5 7}, Chan Young Kang^{5 7}, Mi Young Kim ², Jae Ho Chung ⁸, Jeong-An Gim ⁹, Woochang Hwang ^{10 11}, Yonggoo Kim^{3 4 *}, Song Cheol Kim^{12 *}, Sungho Lee^{8 *}, Junho K Hur ^{11 13 14 *}, Junseok W Hur^{1 5 7 *}

¹GeneCker, Seoul, 04793, South Korea.
²Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, 07061, South Korea.
³Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, 06591, South Korea.
⁴Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 06591, South Korea.
⁵Department of Neurosurgery, College of Medicine, Korea University, Seoul, 02841, South Korea.
⁶Department of Integrated Biomedical and Life Sciences, College of Health Science, Korea University, Seoul, 02841, South Korea.
⁷Center for Genomic Research and Development, Korea University Anam Hospital, Seoul, 02841, South Korea.
⁸Department of Thoracic & Cardiovascular surgery, College of Medicine, Korea University, Seoul, 02841, South Korea.
⁹Department of Medical Science, Soonchunhyang University, Asan-si, Chungcheongnam-do, 31538, South Korea.
¹⁰Department of Pre-Medicine, College of Medicine, Hanyang University, Seoul, 04763, South Korea.
¹¹Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, 04763, South Korea.
¹²Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery, BK21 Project, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, South Korea.
¹³Department of Genetics, College of Medicine, Hanyang University, Seoul, 04763, South Korea.
¹⁴Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, 04763, South Korea.

^†S.Y., J.-S.K., and M.K. contributed equally to this work.

^*Corresponding authors: correspondence to Yonggoo Kim, Song Cheol Kim, Sungho Lee, Junho K Hur or Junseok W Hur

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.

Graphical Abstract. An advanced-fidelity CRISPR nuclease, FnCas9-AF2, is rationally engineered to discriminate single-base mismatches with unprecedented level precision. Integrated into the MUTE-Seq workflow, FnCas9-AF2 depletes wild-type cell-free DNA, thereby exposing rare tumor-derived mutant DNA (a molecular “needle in a haystack”). The method enables ultrasensitive, cost-efficient detection of low-frequency mutations for liquid-biopsy cancer diagnostics, supporting early detection, companion diagnosis, and MRD monitoring.

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