MUTE‐Seq: An Ultrasensitive Method for Detecting Low‐Frequency Mutations in cfDNA With Engineered Advanced‐Fidelity FnCas9
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.
Article Details
Authors (21)
Sunghyeok Ye
GeneCker Seoul 04793 South Korea
Jin‐Soo Kim
Department of Internal Medicine Seoul National University Boramae Medical Center Seoul 07061 South Korea
Myungshin Kim
Department of Laboratory Medicine College of Medicine The Catholic University of Korea Seoul 06591 South Korea
Ki‐Yeon Kim
GeneCker Seoul 04793 South Korea
Yoon‐Ho Won
GeneCker Seoul 04793 South Korea
Taegun Park
GeneCker Seoul 04793 South Korea
Sungjae An
Haerin Jeong
GeneCker Seoul 04793 South Korea
Hee‐Joon Chung
GeneCker Seoul 04793 South Korea
In Seon Lee
GeneCker Seoul 04793 South Korea
Myoung‐Hee Kang
Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea
Chan Young Kang
Department of Neurosurgery College of Medicine Korea University Seoul 02841 South Korea
Mi Young Kim
Department of Internal Medicine Seoul National University Boramae Medical Center Seoul 07061 South Korea
Jae Ho Chung
Jeong‐An Gim
Department of Medical Science Soonchunhyang University Asan‐si Chungcheongnam‐do 31538 South Korea
Woochang Hwang
Department of Pre‐Medicine College of Medicine Hanyang University Seoul 04763 South Korea
Yonggoo Kim
Department of Laboratory Medicine College of Medicine The Catholic University of Korea Seoul 06591 South Korea
Song Cheol Kim
Sungho Lee
Department of Thoracic & Cardiovascular surgery College of Medicine Korea University Seoul 02841 South Korea
Junho K Hur
Hanyang Institute of Bioscience and Biotechnology Hanyang University Seoul 04763 South Korea
Junseok W Hur
GeneCker Seoul 04793 South Korea