2.1.2 Toehold switch and CRISPER-Cas recognition
Cell-free biosensors based on toehold switch and CRISPER-Cas are mainly designed to detect genomic DNA or viral RNA of some pathogens. The toehold switch system is a programmable ribosome regulator, and it consists of two strands of RNA, called a switch or a trigger. The toehold switch sensor controls the translation of genes by triggering the binding of RNA through a trans effect. The switch contains a salient loop structure-forming in the upstream of the gene, which blocks gene translation in cis by blocking the ribosome binding site (RBS) [25]. After binding to the complementary trigger RNA, the sequestration is released, and the gene translation is activated [26]. The target nucleic acid is used as a trigger RNA to activate the expression of the reporter gene in cell-free system. Finally, the detection of the target can be determined directly by the expression of the reporter gene.
The cell-free biosensors based on CRISPER-Cas mainly rely on the cut principle of CRISPER-Cas to identify targets. CFPS system can express many kinds of active CRISPR machinery from plasmids or linear DNAs, and it can further output quantitative dynamics of gene cleavage or repression without the protein purification [27]. According to different Cas effectors (Cas9, Cas12a, and Cas13a), CRISPR biosensor systems have different shear cleavage principles [28]. For example, the CRISPER-Cas13a system can detect analytes that can directly cleave captured targets using the Cas13-crRNA complex [29], and then the lateral cracks of the complex are activated to cleave nearby non-targeted RNA (quenched fluorescent RNA). As a reporter, the quenched fluorescent RNA is cleaved and displays a fluorescence signal to respond to the results.
In addition, toehold switch and CRISPER-Cas can cooperate to detect analytes. For example, Pardee et al . [30] identified two strains by NASBA- CRISPER /Cas9 cleavage method (Fig. 2B). The nucleic acid sequence of a strain can be used as the target sequence. With the participation of reverse transcriptase and T7 RNA polymerase, the proto-spacer adjacent motifs (PAM) and trigger sequence (lacZ trigger) can be introduced in target sequence by isothermal amplification [31]. Cas endonuclease is mediated by sgRNA to recognize the PAM of the target sequence for fixed-point cutting. The uncut sequence contains the trigger RNA, which can be detected by the paper-based toehold switch sensor and finally identified as the strain type by the color change [32].
Nevertheless, there are some challenges to this method. The isothermal amplification process is susceptible to contamination, which could lead to off-target products and false positives. However, this phenomenon can be minimized by using sequentially specific fulcrum switch sensors, and CRISPR-Cas mediated amplification of downstream selection [33]. Multiplexing for CRISPR-Cas systems is also a considerable challenge [34, 35].