Reduced rAAV interference enhances rcAAV detection sensitivity.

Replication-competent adeno-associated virus (rcAAV) content is a crucial contaminant in the process of recombinant adeno-associated virus (rAAV) production in gene therapy products, from preclinical to clinical stages. The gold standard for qPCR-based quantification of rcAAV involves co-infecting HEK293 cells with recombinant adenovirus (rAd) and rAAV samples, followed by amplification and qPCR analysis of both the rcAAV and rAAV genomes. Here, we reported that the presence of large quantity of rAAV interferes with accurate rcAAV detection, leading to false-negative results. In addition, we present a CRISPR-based approach to improve rcAAV detection, where SpCas9 was overexpressed in HEK293 cells and single-guide RNA (sgRNA) was delivered via rAd5 to cleave the rAAV genome. The assay detected as few as 3E2 vector genomes (vg) of rcAAV, whereas the traditional method using the same sample batch failed to detect such low levels. This study not only expands our knowledge of adeno-associated virus (AAV) biology but also highlights a CRISPR-based assay that improves the sensitivity of rcAAV detection.