An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome

Lei Yu; Yong Zhou; Xin-chang Shi; Guang-yu Wang; Zhi-hao Fu; Cheng-gang Liang; Jun-zhi Wang

Molecular Therapy: Methods & Clinical Development (Sep 2024)

An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome

Lei Yu,
Yong Zhou,
Xin-chang Shi,
Guang-yu Wang,
Zhi-hao Fu,
Cheng-gang Liang,
Jun-zhi Wang

Affiliations

Lei Yu: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, Liaoning 110016, P.R. China; State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China
Yong Zhou: State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China
Xin-chang Shi: State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China
Guang-yu Wang: State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China
Zhi-hao Fu: State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China
Cheng-gang Liang: State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China; Corresponding author: Liang Cheng-gang, State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo Street, Daxing District, Beijing 100050, P.R. China.
Jun-zhi Wang: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, Liaoning 110016, P.R. China; State Key Laboratory of Drug Regulatory Science & NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31 Huatuo St, Daxing District, Beijing 100050, P.R. China; Corresponding author: Wang Jun-zhi, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, Liaoning 110016, P.R. China.

Journal volume & issue: Vol. 32, no. 3
p. 101304

Abstract

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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.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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