Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy Manufacturing

Background:

• Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors is crucial for meeting clinical demand.
• A significant challenge is the high production of empty capsids lacking therapeutic genomes.
• Lack of standardized analytical methods complicates accurate quantification of viral genomes and the empty-to-full capsid ratio.

Purpose of the Study:

• To introduce CRISPR diagnostics (CRISPR-Dx) as a robust and rapid method for determining AAV genome titers.
• To develop and validate the CRISPR-AAV Evaluation (CRAAVE) assay for sensitive and precise AAV quantification.
• To demonstrate the potential of CRAAVE for revolutionizing biomanufacturing process analytical technology (PAT).

Main Methods:

• Design and development of the CRISPR-AAV Evaluation (CRAAVE) assay.
• Utilizing CRISPR diagnostics for sensitive detection of AAV genome titers.
• Demonstration of an on-chip CRAAVE assay with lyophilized reagents for simplified use.

Main Results:

• The CRAAVE assay accurately quantified AAV titers with high precision (<3% error) compared to quantitative PCR (qPCR).
• The assay demonstrated a limit of detection as low as 7e7 vg/mL.
• Assay time was reduced to approximately 30 minutes, drastically shortening the analytical workflow.

Conclusions:

• CRISPR diagnostics, exemplified by the CRAAVE assay, provide a promising tool for efficient rAAV genome titer quantification.
• The CRAAVE assay offers high sensitivity, precision, and speed, addressing key challenges in AAV manufacturing.
• This approach has the potential to significantly advance process analytical technology in biomanufacturing.