RAPID-CRISPR: highly sensitive diagnostic assay for detection of PML::RARA isoforms in acute promyelocytic leukemia

Abstract

Acute promyelocytic leukemia (APL), distinguished by the presence of PML::RARA fusion transcript, is a medical emergency because of 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 (<3 hours) detect PML::RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 patients with APL (66/8, retrospective/prospective) and 48 controls were included in the study. We used a DETECTR (DNA endonuclease-targeted CRISPR transreporter) assay to identify the bcr1, bcr2, and bcr3 PML::RARA isoforms. To ensure high specificity, we used PML::RARA-specific loop-mediated isothermal amplification (LAMP) 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 patient samples. Furthermore, 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 1 copy of PML::RARA compared with 10 copies by the gold-standard reverse transcriptase qualitative and quantitative polymerase chain reaction. The nucleic acid extraction-free protocol combined with the 1-step reverse transcriptase 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 underresourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.

Graphical abstract

Figure 1.

Design of RAPID-CRISPR assay. (A) Experimental workflow and timeline of RAPID-CRISPR assay for PML::RARA fusion transcript. (B) Illustration of LAMP primers and crRNA position on bcr1, bcr2, and bcr3 isoforms. sPAM, synthetic protospacer-adjacent motif.

Figure 2.

RAPID-CRISPR for the specific detection of PML::RARA fusion transcript. (A) Isoform-specific RAPID-CRISPR assay in APL and non-APL cell lines. The median with interquartile range is represented; P < .001. (B) Experimental workflow of cDNA dilutions for RAPID-CRISPR and RQ-PCR. (C-D) Quantification of PML::RARA copy number by RQ-PCR in NB4 (blue) and UF-1 (pink) cDNA dilutions. (E-F) Quantification of fluorescence by RAPID-CRISPR using cDNA dilutions in NB4 (blue) and UF-1 (pink). (G) LOD of PML::RARA using cDNA from an APL case.

Figure 3.

Validation of RAPID-CRISPR assay using clinical samples. (A) Measurement of fluorescence using isoform-specific crRNA in APL cases (n = 66) and controls (n = 43) by RAPID-CRISPR. The result is represented as a median with an interquartile range; P < .0001. (B) Fluorescence data of bcr1, bcr2, and bcr3 isoforms of PML::RARA by RAPID-CRISPR. A median with an interquartile range represents the data P = ns (nonsignificant). (C) Fluorescence data of APL cases from the PB (n = 54) and BM (n = 12). The data are represented as a median with interquartile range; P = ns. (D) ROC curve to estimate the clinical sensitivity and specificity of the RAPID-CRISPR assay with APL (n = 66) and control (n = 43) cases. Area under the curve (AUC) = 1.000; 95% confidence interval (CI), 1.000-1.000. (E) Illustration of blinded validation of RAPID-CRISPR in APL (n = 9) and control (n = 9) cases. (F) ROC curve to estimate the clinical sensitivity and specificity of the RAPID-CRISPR assay in a blinded validation experiment. AUC = 1.000; 95% CI, 1.000-1.000. (G) Measurement of fluorescence between MRD⁻ (n = 6) and MRD⁺ (n = 6) APL cases by RAPID-CRISPR assay. The data are represented as a median with interquartile range; P < .01. (H) LF-based readouts of RAPID-CRISPR assay for PML::RARA detection using 5 APL cases and 5 controls.

Figure 4.

PML::RARA detection by RAPID-CRISPR assay using RT-LAMP to expedite the turnaround time of APL diagnosis. (A) Optimization of RT-LAMP using PB of 2 healthy individuals (H1 and H2) served as a control (no template control [NTC]). (B) Quantification of fluorescence of GUSβ by RAPID-CRISPR assay using 1-step RT-LAMP in control samples. (C) Quantification of fluorescence of PML::RARA by RAPID-CRISPR assay using RT-LAMP in APL cases (n = 8) and control (n = 5). The results are represented as a mean ± standard deviation; P < .0001. (D) LF-based readouts of 4 APL cases and 4 controls by RAPID-CRISPR assay using RT-LAMP.