Toward point-of-care and amplification-free detection of human cytomegalovirus using CRISPR-Cas12a

Human cytomegalovirus (hCMV) is a herpesvirus that establishes lifelong latency in myeloid cells, posing health concerns particularly in fetal development and in immunocompromised individuals. Point-of-care (PoC) detection of hCMV DNA in liquid biopsies supports timely diagnosis and proper mitigation. However, ultra-low concentrations and high fragmentation rates, challenge primer-based preamplification methods. We present a proof-of-concept amplification-free CRISPR-based assay, exploiting the inherent specificity and signal-amplification of Cas12a and improving signal using a combinatorial approach. Optimizing Cas12a's trans -cleavage activity and multiplexing hCMV loci, significantly increased detection sensitivity in-bulk. Additionally, we found that AsCas12a trans -cleaves cytosine-rich reporters 4× more efficiently than conventional probes, further improving assay kinetics to reach a femtomolar limit of detection. Translating these optimizations to a microfluidic assay enables sensitive detection even if additional measures may be needed for quantitative, single molecule measurements. Our assay opens avenues toward PoC detection in low-resource settings, supporting effective and affordable infection management.