Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice

文献类型： 外文期刊

第一作者： Su, Zhixun

作者： Su, Zhixun;Zeng, Xiaoqun;Su, Zhixun;Wang, Xiaofu;Chen, Xiaoyun;Ding, Lin;Xu, Junfeng;Peng, Cheng

作者机构：

关键词： Gene editing; Visual detection; CRISPR/SpRY; In vitro cleavage; RPA

期刊名称：ANALYTICA CHIMICA ACTA （ 影响因子：6.2； 五年影响因子：5.9 ）

ISSN： 0003-2670

年卷期： 2024 年 1303 卷

页码：

收录情况： SCI

摘要： The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/ SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.

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