MUTE-Seq: An Ultrasensitive Method for Detecting Low-Frequency Mutations in ctDNA with Engineered Advanced-Fidelity FnCas9

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. We developed MUTE-Seq by applying FnCas9-AF2 for enrichment of mutant DNA without sequence limitations via the exclusive cleavage. The depletion of perfectly matched wild-type DNA targets offered a sensitive detection method for 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). Multiplexed MUTE-Seq (mMUTE-Seq) resulted in a significant improvement in the sensitivity of simultaneous mutant detection and was also effective for stage I NSCLC patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9-AF2-based MUTE-Seq method could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.