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].