Rapid Adeno-Associated Virus Genome Quantification with Amplification-Free CRISPR-Cas12a

Abstract

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision ( View larger version (24K): org.highwire.dtl.DTLVardef@1bfce18org.highwire.dtl.DTLVardef@17393c9org.highwire.dtl.DTLVardef@eb7c1forg.highwire.dtl.DTLVardef@2203cd_HPS_FORMAT_FIGEXP M_FIG C_FIG