A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice

Abstract

Abstract Background In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing. Objective We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products. Methods In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods. Results The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products. Conclusion We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice. Highlights The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.