Högskolan i Skövde

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor long-term survival (only ~20–30% of patients live five years even with intensive therapy). Current diagnosis relies on sequencing, but this can take days to weeks, delaying targeted treatment. CRISPR-based tests promise much faster results (within a couple of hours). In this project, a CRISPR diagnostics (CRISPR-Dx) assay was developed to detect two key AML mutations – the NPM1-Type A (4-base insertion) and the IDH1-R132C (single-nucleotide) mutations. Synthetic guide RNAs (crRNAs) were designed for each target and assembled with Cas12 nucleases (especially an enhanced AsCas12a, “enAsCas12a”) to form RNP (ribonucleoprotein; crRNA plus Cas protein) complexes. The assay used a fluorophore-quencher (FQ) reporter that emits fluorescence only when cleaved by the activated Cas enzyme. Real-time fluorescence (FQ) assays were performed, and end-point DNA cleavage assays were conducted. Additionally, an amplicon sequencing (NGS) pipeline was built to genotype the same samples. enAsCas12a produced a strong, specific signal for the NPM1 insertion, reliably distinguishing mutant from wild-type, as little as 0.25% presence of the mutant allele was detectable, and with significant specificity. In contrast, enAsCas12a failed to discriminate the single-base IDH1-R132C mutation from wild-type with crRNAs. Cas14a (Cas12f) and a high-fidelity Cas12 variant (hfCas12Max) showed negligible activity and/or specificity under the experiment’s conditions. By comparison, the NGS genotyping pipeline correctly identified the mutation status of all AML and control samples. In summary, this project demonstrates that a CRISPR-Cas12a assay can feasibly detect the NPM1-Type A indel mutation in AML, but that single-nucleotide variant detection (IDH1-R132C) remains challenging. These findings provide proof-of-concept that CRISPR-Dx could shorten AML mutation testing, while highlighting the need for optimized guide design and chemistry to handle subtle mutations.