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We engineered a highly precise advanced\u2010fidelity FnCas9 variant, named FnCas9\u2010AF2, to effectively discriminate single\u2010base mismatches at all positions of the single guide RNA (sgRNA) target sequences. FnCas9\u2010AF2 exhibited significantly lower off\u2010target effects compared to existing high\u2010fidelity CRISPR\u2010Cas9 variants. MUTE\u2010Seq leverages FnCas9\u2010AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild\u2010type DNA, allowing for sensitive detection of low\u2010frequency cancer\u2010associated mutant alleles. MUTE\u2010Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE\u2010Seq was applied in a multiplexed manner on cell\u2010free DNA (cfDNA) from patients diagnosed with non\u2010small 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\u2010stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9\u2010AF2\u2010based MUTE\u2010Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.<\/jats:p>","DOI":"10.1002\/adma.202505208","type":"journal-article","created":{"date-parts":[[2025,8,22]],"date-time":"2025-08-22T05:30:46Z","timestamp":1755840646000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["MUTE\u2010Seq: An Ultrasensitive Method for Detecting Low\u2010Frequency Mutations in cfDNA With Engineered Advanced\u2010Fidelity FnCas9"],"prefix":"10.1002","volume":"37","author":[{"given":"Sunghyeok","family":"Ye","sequence":"first","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin\u2010Soo","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine Seoul National University Boramae Medical Center Seoul 07061 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myungshin","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine College of Medicine The Catholic University of Korea Seoul 06591 South Korea"},{"name":"Catholic Genetic Laboratory Center Seoul St. Mary's Hospital College of Medicine The Catholic University of Korea Seoul 06591 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ki\u2010Yeon","family":"Kim","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoon\u2010Ho","family":"Won","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taegun","family":"Park","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"},{"name":"Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea"},{"name":"Department of Integrated Biomedical and Life Sciences College of Health Science Korea University Seoul 02841 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sungjae","family":"An","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haerin","family":"Jeong","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hee\u2010Joon","family":"Chung","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"In Seon","family":"Lee","sequence":"additional","affiliation":[{"name":"GeneCker Seoul 04793 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myoung\u2010Hee","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea"},{"name":"Center for Genomic Research and Development Korea University Anam Hospital Seoul 02841 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chan Young","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea"},{"name":"Center for Genomic Research and Development Korea University Anam Hospital Seoul 02841 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mi Young","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine Seoul National University Boramae Medical Center Seoul 07061 South Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jae Ho","family":"Chung","sequence":"additional","affiliation":[{"name":"Department of Thoracic &amp; 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