CRISPR-Cas12a-Assisted Multicolor Biosensor for Semiquantitative Point-of-Use Testing of the Nopaline Synthase Terminator in Genetically Modified Crops by Unaided Eyes
Di Huang, Jiajie Qian, Zhuwei Shi, Jiarun Zhao, Mengjun Fang, Zhinan Xu
Abstract
Existing methods of detecting foreign genes and their expression products from genetically modified organisms (GMOs) suffer from the requirement of professional equipment and skillful operators. The same problem stays for the CRISPR-Cas12a system, although it has been emerging as a powerful tool for nucleic acid detection due to its remarkable sensitivity and specificity. In this report, a portable platform for the visible detection of GMOs based on CRISPR-Cas12a was established, which relies on a color change of gold nanorods (GNRs) caused by the invertase-glucose oxidase cascade reaction and the Fenton reaction for signal readout. A nopaline synthase (NOS) terminator was employed as a model target commonly existing in foreign genes of GMOs. With the help of recombinase-aided amplification, this platform achieved comparable sensitivity of DNA targets (1 aM) with that of a fluorescence reporting assay. As low as 0.1 wt % genetically modified (GM) content in Bt-11 maize was visually observed by unaided eyes, and the semiquantitation of GM ingredients can be obtained within the range of 0.1 to 40 wt % through the absorption measurement of GNRs. Furthermore, five real samples were tested by our method, and the results indicated that the GM ingredient percentages of GMO samples were 2.24 and 24.08 wt %, respectively, while the other three samples were GMO-free. With the advantages of a simple procedure, no need for large or professional instruments, high sensitivity, and selectivity, this platform is expected to provide reasonable technical support for the safe supervision of GMOs.