RAPID-CRISPR: Highly Sensitive Diagnostic Assay for Detection of PML-RARA Isoforms in Acute Promyelocytic Leukemia

• RAPID-CRISPR assay provides swift diagnosis of APL that facilitates timely therapeutic intervention minimizing the risk of early mortality

• Designed for resource-limited settings, this assay features an easy-to-read format with 100% sensitivity and 100% specificity

Acute promyelocytic leukemia (APL), distinguished by the presence of PML-RARA fusion transcript, is a medical emergency due to its high early death rate, which is preventable when diagnosed early. Current diagnostic methods are precise and reliable but are time-intensive, require sophisticated instruments and analytical expertise. This study has Redefined APL IDentification by CRISPR system (RAPID-CRISPR) to rapidly (<3hrs) detect PML-RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 APL patients (66/8 retrospective/prospective) and 48 controls were included in the study. We utilized DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) assay to identify the bcr1, bcr2, and bcr3 PML-RARA isoforms. To ensure high specificity, we used PML-RARA-specific LAMP (loop-mediated isothermal amplification) primers, synthetic protospacer adjacent motif sites, and isoform-specific crispr RNAs. RAPID-CRISPR recognized APL with 100% sensitivity and 100% specificity in an ambispective cohort of patients' samples. Further, our blinded validation approach to detect PML-RARA in an unbiased manner provides an additional layer in the diagnostic precision of APL. RAPID-CRISPR demonstrated superior sensitivity, detecting as few as one copy of PML-RARA compared to 10 copies by the gold standard RQ-PCR. The nucleic acid extraction-free protocol combined with the one-step RT-LAMP-based DETECTR followed by lateral flow readout makes the RAPID-CRISPR assay suitable for diagnosing APL in point-of-care settings. This simple, cost-effective tool, with its easy-to-read format, is particularly valuable in under-resourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.