Sprecher
Beschreibung
The programmability of CRISPR-based techniques, combined with the advantages of nucleic acid nanotechnology, has enabled the development of innovative diagnostic tools.
Here we present a two-step CRISPR-based immunoassay, named MAIGRET (Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription). This platform enables the sensitive and versatile detection of specific antibodies and other proteins by integrating the advantages of CRISPR-based sensing with cell-free transcription systems.
In the first step of the assay, the target antibody binds to a pair of antigen-conjugated DNA strands, inducing their co-localization and the formation of a bimolecular complex. This complex hybridizes to the single-stranded region of an inactive synthetic template that encodes the CRISPR RNA guide strand (crRNA) specific to the Cas12a enzyme. Only upon formation of this complex the synthetic template is activated, allowing the cell-free transcription of the crRNA strand. In the second step, an aliquot of the first reaction is transferred into a mixture containing Cas12a, its double-stranded DNA activator, and a DNA hairpin reporter labeled with a fluorophore-quencher pair. The crRNA produced in the first step activates Cas12a, triggering its collateral cleavage activity and resulting in the reporter cleavage and a measurable fluorescence signal.
MAIGRET achieves highly sensitive (detection limits in the low picomolar range), specific (no signal in the presence of non-target antibodies), and selective (effective even in complex samples such as 50% blood serum) detection of target proteins. Thanks to its programmable design, the platform can be tailored for different targets, either in direct or competitive assay formats, and supports multiplexed detection using orthogonal CRISPR enzymes (e.g., Cas13), each coupled with a distinct, antibody-responsive transcriptional template. Considering the versatility of MAIGRET, we are currently developing an adaptation with a colorimetric readout by employing the collateral cleavage of a horseradish peroxidase (HRP)-DNA strand anchored on the magnetic beads as reporter system.
