A Synthetic Light-switchable System based on CRISPR Cas13a Regulates the Expression of LncRNA MALAT1 and Affects the Malignant Phenotype of Bladder Cancer Cells.

Abstract

DNA sequences drive their various functions through post-transcriptional processes, using mRNA or lncRNA (long non-coding RNA), and this accommodates the gene network by using various RNA types. However, the tools necessary to regulate RNA molecules are few. Likewise, RNA knockdown techniques that can be artificially controlled have not been extensively explored. Here, we investigated a novel light-inducible synthetic system based on CRISPR-Cas13a that can be used for RNA knockdown and binding in cancer cells. Based on the techniques of synthetic molecular biology, we constructed a light sensor, which efficiently induced Cas13a protein expression after blue light illumination. We also chose a lncRNA, Metastasis-associated Lung Adenocarcinoma Transcript 1 (MALAT1), as the functional target and detected it in bladder cancer 5637 and T24 cells in order to demonstrate the application of our synthetic system. Fluorescence reporter assays and real-time quantitative PCR (qRT-PCR) were used to detect the expression of the target gene. Phenotypic experiments were also used to test the effects of our synthetic system in bladder cancers. The results showed that our synthetic light-switchable system could inhibit the expression of MALAT1, and the fluorescence activity of enhanced green fluorescent protein. Our novel system provides a new technique to study RNA functions in gene networks and for precise tumor treatments.