Label-Free Detection of Transgenic Crops Using an Isothermal Amplification Reporting CRISPR/Cas12 Assay

Abstract

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars (“Bt11” and “MON89034”) from nontransgenic corn cultivars (“yellow”, “shenyu”, “xianyu”, and “jingke”). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

“…CRISPR/Cas12a, an RNA-guided DNase, demonstrates ssDNase ability (also called collateral effect or collateral cleavage) when the Cas12a/crRNA complex is activated by the target dsDNA. − In the CRISPR/Cas12a detection system, ssDNA is usually dual-labeled with a fluorophore and quencher so that the dsDNA input can be transformed into the measurable fluorescent output. ,, CRISPR/Cas12a is advantageous for being both temperature-resilient and highly specific . Cas12a can be activated even at room temperature and performs low mismatch tolerance. , These features enable the CRISPR/Cas12a system to be an efficient and programable biosensing platform. − However, the CRISPR/Cas12a system alone is challenged for its limited sensitivity. , A typical CRISPR/Cas12a system can hardly provide detectable signals when the concentration of the dsDNA substrate is lower than 100 pM . To compensate for such limitations, the standard strategy is priorly using nucleic-acid amplification to increase the quantity of the target dsDNA substrate in the CRISPR/Cas12a system.…”

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…In addition, a series of visual equipment and analyses were introduced to avoid the dependency on laboratory instruments. A label-free G-rich DNAzyme (Zhu et al, 2022), UV light (Lee & Oh, 2022), and so on were utilized as the reporting signals. Yin, Duan, et al (2021) integrated a smartphone for ultrasensitive detection of pathogenic bacteria.…”

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice

“…A clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR/Cas) system has been proven powerful in developing assays for nucleic acid biomarkers. The RNA-guided recognition enables the CRISPR tools to be highly specific, and the multiple turnover of collateral cleavage provides signal amplification that contributes to sensitivity improvement. − CRISPR-based nucleic acid detection has been widely applied in monitoring environmental and food safety, mainly targeting pathogens and genetically modified organisms . To enhance the sensitivity, the CRISPR technologies are generally combined with isothermal amplification strategies, such as recombinase polymerase amplification and loop-mediated isothermal amplification .…”

“…14−17 CRISPR-based nucleic acid detection has been widely applied in monitoring environmental and food safety, mainly targeting pathogens 18 and genetically modified organisms. 19 To enhance the sensitivity, the CRISPR technologies are generally combined with isothermal amplification strategies, such as recombinase polymerase amplification 18 and loop-mediated isothermal amplification. 20 Using CRISPR tools to detect non-nucleic acid biomarkers, however, requires compatible biorecognition and signal transduction elements.…”