An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome
Brief intro:
- Author: Lei Yu, Yong Zhou, Xin-chang Shi, Guang-yu Wang, Zhi-hao Fu, Cheng-gang Liang, Jun-zhi Wang
- Journal: Molecular Therapy
- Doi: https://www.doi.org/10.1016/j.omtm.2024.101304
- Publication Date: 2024 Jul 21
Products/Services used in the paper
Quotation shows PackGene:AAV5-IN, the in-house reference produced by Packgene Biotech (Guangzhou, China), was purified using affinity chromatography and iodixanol gradient ultracentrifugation.
Research Field:CRISPR-Cas9
AAV Serotype:AAV5
Abstract
The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying the rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p < 0.05, t test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.
About PackGene
PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
