Highly Sensitive and Rapid Diagnosis of Mutations in Hematological Malignancies By CRISPR Technology

With the rapid development of genomics and molecular diagnosis technologies, the treatment of hematological malignancies has entered the era of precision medicine, where individualized treatment is provided based on the unique genetic profile of each patient. Commonly-used techniques for gene mutation detection include polymerase chain reaction (PCR), sequencing approaches and real-time fluorescence quantitative PCR. However, these methods have drawbacks in sensitivity, specificity, or speed. Here, we established a mutation detection method based on CRISPR technology, which can achieve a highly sensitive, specific, and rapid diagnosis. Through a rapid release of genomic DNA, isothermal amplification, mutation enrichment, and Cas12a fluorescence reaction, this method can complete the whole diagnosis process from drawing blood to obtaining the results within one hour, without any large instruments or expensive reagents. Taking the detection of FLT3 gene D835Y/V/H/F drug-resistant mutations as an example, the sensitivity can reach 0.001%, which means 10 copies of mutation template can be detected from 999,990 copies of wild-type template. Then we used this method to detect 112 patients with acute myeloid leukemia, resulting in 11 FLT3-D835Y/V/H/F positive cases, which were verified by next-generation sequencing while missed by Sanger sequencing. We also applied this method to detect other common mutations in hematological malignancies, including NRAS gene G12D, IDH2 gene R172K, and JAK2 gene V617F. These results fully proved the high sensitivity, specificity, efficiency and universality of this method. Thus it has the potential to become a novel molecular diagnostic technology, and help point-of-care, early screening, prognosis determination and medication guidance in hematological malignancies.