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 (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).Graphical Abstract

A Ratiometric Nonfluorescent CRISPR Assay Utilizing Cas12a-Induced Plasmid Supercoil Relaxation

“…The gel image in Figure S5 confirmed that supercoil relaxation of ΦX174 is equally valid for dsDNA targets. Then, we chose two real-life viral genomes AAV 78 and HPV 16 47 and used their genetic markers (see the sequences in Table S1 ) as the model targets to challenge rCRISPR. The gel image in Figure 7 shows that DNA supercoil relaxation mechanism is universally effective for both targets.…”