CRISPR/Cas12a-drived fluorescent and electrochemical signal-off/on dual-mode biosensors for ultrasensitive detection of EGFR 19del mutation

The epidermal growth factor receptor (EGFR), a member of the epidermal growth factor receptor (HER) family, is closely related to the activation of cell signaling pathways, including cell proliferation and apoptosis protection [1], [2]. Studies have been proved that EGFR is over-expressed in various tumors, especially in lung adenocarcinoma. This abnormal gene expression is primarily caused by EGFR mutations in exons 18–21 of the tyrosine kinase region, which leads to sustained activation of tyrosine kinase and drives the development of non-small cell lung cancer (NSCLC) [3]. Among the EGFR mutations, EGFR 19 deletion (EGFR 19del) and EGFR L858R point mutation cover 90% [4]. The EGFR 19del occurs most frequently, accounting for approximately 48% [5]. Therefore, accurate and sensitive identification of EGFR mutation status is a prerequisite for cancer early diagnosis and the guidance of tumor targeting drugs.

Currently, a variety of molecular biological techniques have been developed for identifying of EGFR mutations. The next-generation sequencing (NGS) is the most common and direct method for EGFR mutation detection [6]. However, its widespread application is limited by high cost, poor sensitivity and time consuming [7]. Although the polymerase chain reaction (PCR) based diagnostic assays, such as Cobas TaqMan-based PCR [8] and droplet digital PCR (ddPCR), [9] enable the quantification of trace amounts of DNA, these methods require circulating heating equipment, so they are not suitable for point-of-care testing [10]. Alternatively, some isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP) [11], primer exchange reaction (PER) [12], and strand displacement reaction (SDA) [13], have received extensive attention. Isothermal amplification has the advantages of rapid reaction, high amplification efficiency and no special equipment [14], [15], [16]. However, in actual biological samples, the target sequence is mostly in a non-mutated state. Therefore, a protocol that combines signal amplification is critical for the high sensitivity and accuracy of mutation site detection.

Recent studies have shown that molecular diagnostic systems based on CRISPR/Cas technology have been incorporated into practice in many isothermal systems. As expected, a wide variety of sensing platforms have been gradually established, such as SHELOCK (Cas13a) [17], DETECR (Cas12a) [18], CAS-EXPAR (Cas9) [19] and E-CRISPR (Cas12a) [20], which have enabled highly sensitive analysis of nucleic acids, proteins, and other substances [21], [22], [23]. The main function of CRISPR/Cas technology is to further amplify the signal after nucleic acid amplification, and then carry out signal conversion and output [24], [25]. Specifically, some Cas proteins assembled with crRNA are used to recognize the amplification product, activating the cleavage activity of these Cas proteins, and then act on the substrate probe that can be designed for different signal output modes [26]. According to the different signal input mode, fluorescence and electrochemical sensing are the two commonly used signal output modes based on the advantages of high sensitivity, selectivity, and stability [27], [28], [29], [30], [31]. Compared with single signal output, multimodal sensing strategies rely on different response mechanisms and own inherent characteristics of each response mode. Therefore, the test results can be verified by different modes, and the accuracy and sensitivity of the analysis can be improved by data complementarity and self-calibration [32], [33]. Yu et al. integrated exponential amplification reaction (EXPAR) and CRISPR/Cas12a to develop fluorescence and electrochemical sensors for m6A detection. This protocol can identify methylation targets with abundance as low as 1% [34]. Therefore, sensors based on dual-signal output patterns can achieve accurate and sensitive quantification of analytes relevant to early clinical diagnosis and disease treatment.

In this work, we combine rolling-circle strand displacement amplification (RC-SDA) with CRISPR/Cas12a system to develop fluorescent and electrochemical signal-off/on dual-mode biosensors for detection of EGFR 19del mutation. By introducing a padlock probe for target recognition, the probe can form a closed circle in the presence of normal EGFR 19 gene. The closed padlock probe serves as a template to trigger the RC-SDA reaction. Since the template contains two endonuclease sites, a primer recognition region, and an amplification region, the rolling-circle strand displacement amplification with high efficiency can be implemented. The amplification products are then applied to the Cas12-based fluorescence and electrochemical sensing platform. CRISRP/Cas12a can specifically recognize the product for further signal amplification and output. Experimental results show that the Cas12-based fluorescence biosensor can detect as low as 18.62 fM. Moreover, it exhibits the ability to identify 0.5% EGFR 19del mutations in the presence of a large amount of wild-type EGFR 19. For electrochemical sensing, the limit of detection is 0.13 fM and 0.1% of mutation target can be identified. It is found that Cas12-based electrochemical sensing shows better detection performance, which is attributed to the inherent advantages of electrochemical biosensors with high selectivity and high sensitivity. Furthermore, the biosensors successfully detect in-frame deletions of EGFR 19 in cancer cells.

Read more here: Source link