17454 Leveraging CRISPR-Cas12a for the detection of human T-Cell leukemia virus type 1

Human T-cell leukemia virus type 1 (HTLV-1) is a potent carcinogenic oncovirus that, while asymptomatic in the majority, has the potential to cause devastating complications such as adult T-cell leukemia/lymphoma (ATLL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Despite its high morbidity and global burden, poor access to current testing modalities limits HTLV-1 testing in endemic regions. Here we perform the pre-clinical, proof-of-concept experiments necessary for the development of a sensitive, specific, and low-cost CRISPR-based screening test for HTLV-1. We took advantage of recent reports describing the ability of Cas12a enzymes and guide RNAs (gRNAs) to act as molecular sensors in field-deployable viral diagnostic assays. Upon specific recognition of the gRNA-complementary DNA sequence, eg HTLV-1 proviral DNA, Cas12a/gRNA complexes can catalyze nonspecific trans-cleavage of a single-stranded DNA (ssDNA) reporter, providing an amplified signal of the target recognition event (2). We designed and synthesized multiple HTLV-1-specific Cas12a gRNAs targeted to conserved regions of the HTLV-1 genome. We also designed a custom ssDNA reporter molecule that could be used to measure target-induced Cas12a trans-cleavage activity by quantitative polymerase chain reaction (qPCR). Upon detection of HTLV-1-specific targets in vitro, Cas12/gRNA complexes demonstrated concentration-dependent cleavage of ssDNA. Our assay was able to detect 36 femtomoles of HTLV-1 target DNA and lays the foundation for future efforts to develop a robust, point-of-care CRISPR-based HTLV-1 diagnostic test that could potentially transform the detection and management of HTLV-1 infection.