**3.7 CRISPR-mediated assays**

CRISPR/Cas9 system is a new gene-editing technology based on the bacterial adaptive immune defense system. It can insert or delete genes accurately to knock out target genes [47]. CRISPR/Cas9 system consists of CRISPR RNA, transactivating crRNA, and endonuclease Cas9. Cas9 is specific to the protospacer adjacent

#### *Novel Biosensing Strategies for the* in Vivo *Detection of microRNA DOI: http://dx.doi.org/10.5772/intechopen.93937*

motif with the guidance of crRNA and tracrRNA to form RNA–DNA complex. Cas9 can cut double-stranded DNA to complete gene editing.

For the convenience of operation, scientists fused the mature tracrRNA-crRNA dual structure into a sgRNA, its 5′ strand sequence is complementary to the target miRNA, and the 3′ stranded structure could bind to Cas9. Therefore, only one sgRNA needs to be designed to edit the related genes. This method has some advantages such as simple operation, high efficiency, low cost, and no introduction of foreign genes. So far, it has become the most popular gene-editing technology. Similarly, this method has been applied to the detection of miRNA.

Wang *et al.* [48] constructed a CRISPR–Cas9 sensing platform based on a miRNA-mediated sgRNA releasing strategy. The sensing approach successfully realized miRNA imaging and cell-specific regulation of the CRISPR gene editing system. The sensing system was divided into two parts. One part was composed of Cas9 or Cas9 mutant fusion protein to edit or inhibit gene expression, the other was sgRNA with miRNA binding site without activity, mediating the binding of Cas9 protein in a specific position of the genome. As a result, only specific miRNA could finish cleavage reaction, producing mature sgRNA, and starting CRISPR system. The system could sense two kinds of miRNAs in the same cell and turn on red or green fluorescence respectively. It was proved that the detection platform could accomplish precise regulation of different gene sites by multiple miRNAs, and provided new ideas for other gene therapy technologies.
