Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

Abstract

Introduction: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation.

Objective: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection.

Methods: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically.

Results: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability.

Conclusion: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

Keywords: Dual CRISPR; Genetically Modified (GM) detection; Multiplexed lateral flow strips; One-pot reaction; Recombinase polymerase amplification (RPA).

Graphical abstract

Fig. 1

The principle of MR-DCA for detecting CaMV35S and NOS. A: Schematic of the MR-DCA process. B: Principle for the fluorescence system for MR-DCA. C: Principle of the lateral flow strip system for MR-DCA. NC: Negative controls.

Fig. 2

Feasibility validation of Cas13a and Cas12a detection systems. A: RPA products containing the T7 promoter and endpoint fluorescence image of the Cas13a system after the trans-cleavage reaction. M: 100–1000 bp. Blue light: λ_ex = 494 nm, λ_em = 518 nm. Green light: λ_ex = 535 nm, λ_em = 556 nm. Red box: high fluorescence intensities. B: Fluorescence intensity of CaMV35S and NOS detected by the Cas13a system. (ns: p > 0.05). C: RPA amplification product and endpoint fluorescence image of the Cas12a system after the trans-cleavage reaction. D: Fluorescence intensity of CaMV35S and NOS detected by the Cas12a system. *, p < 0.05. Error bars presented as mean ± S.D. (n = 3). NC: Negative controls. E: Trans-cleavage specificity of the Cas13a system. F: Trans-cleavage specificity of the Cas12a system. GM rice: The KF6 transformant, containing CaMV35S and NOS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Verification of the feasibility of MR-DCA. A: Operational procedures for System 1 of MR-DCA. B: RPA products of CaMV35S and NOS. C: Fluorescence intensity of CaMV35S and NOS detected by MR-DCA. Error bars presented as mean ± S.D. (n = 3). D: Endpoint fluorescence image of CaMV35S and NOS detected by MR-DCA. NC: Negative controls.

Fig. 4

Time and sensitivity measurement for MR-DCA (System 1). A: Fluorescence intensity for different reaction times for CaMV35S detection (CRISPR reaction for 20 min and 10–30 min for the RPA reaction time). B: Fluorescence intensity for different reaction times for NOS detection. C: Endpoint fluorescence images for different RPA reaction times, after a 20 min dual CRISPR reaction (30 s per cycle). D: Fluorescence intensity vs copy number for MR-DCA. Error bars presented as mean ± S.D. (n = 3). E: Linear relationship between fluorescence intensity and copy number of NOS and CaMV35S, Lg C: Logarithm of the number of copies. F: Endpoint fluorescence image for different copy numbers for MR-DCA. NC: Negative controls.

Fig. 5

Time and sensitivity measurement for MR-DCA (System 2). A: Operational procedures for System 2 of MR-DCA. B: Lateral flow strip results for different reaction times (CRISPR reaction of 20 min, 10 min-30 min for the RPA reaction time). C: Line intensities were quantified using Image J and drawn as curves. D: Lateral flow strip results for different NOS and CaMV35S copy numbers. 1: 2 × 10⁵ copies; 2: 2 × 10⁴ copies; 3: 2 × 10³ copies; 4: 1 × 10³ copies; 5: 2 × 10² copies; 6: 2 × 10¹ copies, 7: NC: Negative controls. E: Line intensities were quantified using Image J and drawn as 3D curves. 1: 2 × 10⁵ copies; 2: 2 × 10⁴ copies; 3: 2 × 10³ copies; 4: 1 × 10³ copies; 5: 2 × 10² copies; 6: 2 × 10¹ copies, 7: NC: Negative controls.

Fig. 6

Results of 24 crop samples tested with MR-DCA. A: Endpoint fluorescence image for 24 crop samples. (1, 2, 15, 16, 21 and 22 were independent individual assays for herbicide-tolerant transgenic maize, MON810, which contained the CaMV35S promoter. 7, 8, 23 and 24 were independent individual assays for insect-resistant transgenic rice, TT51-1, which contained the NOS terminator. 3, 4, 11, 12, 13, 14, 17 and 18 were independent individual assays for herbicide-tolerant transgenic soybean, ZUTS-33, which contained the CaMV35S promoter and NOS terminator.) B: Lateral flow strip results for 24 crop samples. T1 line: NOS, T2 line: CaMV35S. C: Heat map analysis of fluorescence intensity. D: Heat map analysis of lateral flow line intensity after quantification using Image J (“+”, positive; “−”, negative). E: Heat map analysis of Ct values.