Adaptable, quantitative CRISPR/Cas12a-based assay for cytomegalovirus DNA in infant saliva

Abstract Congenital cytomegalovirus (CMV) infection is the leading non-genetic cause of infant hearing loss worldwide, and a significant cause of neurodevelopmental disabilities. Reliance on polymerase chain reaction (PCR) for CMV DNA testing hampers diagnostic and research efforts in low-resource settings and universal screening implementation in high-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) detection and recombinase polymerase amplification (RPA) can be used together for low-cost viral detection. Here we describe an adaptable RPA-Cas12a assay for CMV DNA quantification based on the WHO international standard. Adequate quantification accuracy was achieved with contrived CMV samples but performance with Sierra Leonean infant saliva remains suboptimal. While improved quantification accuracy will require further optimization, our assay achieves screening test requirements, including > 80% sensitivity/specificity, quicker and more economically than PCR. This work highlights RPA-Cas12a-based assays for DNA quantification and suggests a path towards increased congenital CMV screening using PCR and RPA-Cas12a synergistically.