Qualitative and Quantitative Detection of CRISPR-Associated <i>Cas</i> Gene in Gene-Edited Foods
Lin Ding,
Xiaoli Xu,
Xiaofu Wang,
Xiaoyun Chen,
Yuwen Lu,
Junfeng Xu,
Cheng Peng
Affiliations
Lin Ding
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Xiaoli Xu
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Xiaofu Wang
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Xiaoyun Chen
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Yuwen Lu
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of MARA and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China
Junfeng Xu
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Cheng Peng
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the Cas gene. In the present study, the primers and probes were designed and screened for Cas12a (Cpf1), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting Cpf1 in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing Cpf1 DNA, while the detection rate of other samples without Cpf1 was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for Cpf1 are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future.
