Engineered Cas12a-based one-tube detection of DNMT3A R882 H/C mutation in acute myeloid leukemia

Abstract

Advances in sequencing technologies have identified numerous genetic alterations associated with acute myeloid leukemia (AML), many of which play critical roles in diagnosis, classification, and prognosis. Among these, mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are particularly prevalent, with the R882H and R882C variants being the most common. Accurate and sensitive detection of DNMT3A mutations is crucial for prognosis, treatment guidance, and early intervention in AML. However, existing detection methods often fail to achieve an optimal balance among sensitivity, turnaround time, and operational simplicity. To address this limitation, we aimed to develop a rapid and highly sensitive method for detecting DNMT3A mutations. The CRISPR/Cas12a system shows promise for genetic detection due to its high sensitivity and single-base specificity. Here, we established a Cas12a-based one-tube assay for the detection of DNMT3A R882 H/C mutations. We utilized the mismatch tolerance of enAsU-R Cas12a to design crRNA for DNMT3A R882 H/C mutation and integrated CRISPR/Cas12a system with ERA. The entire detection process can be completed within 1 h at 37 °C. The optimized detection system demonstrated a sensitivity of 0.1 % when analyzing genomic DNA. To validate its clinical applicability, we tested samples from 49 AML patients and successfully identified all DNMT3A R882H/C-positive cases, including one with a mutation rate as low as 0.24 %. These results highlight the potential of our Cas12a-based one-tube detection system as a rapid, sensitive, and cost-effective method for detecting DNMT3A R882 H/C mutation. This approach could serve as a valuable tool for both diagnostic and therapeutic monitoring.