Design, Modeling and Computational Analysis of crRNA to Regulate MetastamiR-10b and MetastamiR-126 in Post-transcriptional Level by CRISPR-C2c2 (Cas13a) Technique

Metastasis is one the most important causes of mortality in cancer patients. Recent studies have shown the metastatic potential of a specific group of microRNAs called metastamirs.  miR-126 is shown to be correlated with the colorectal liver metastasis. Also, overexpression of miR-10b has been reported in metastatic breast cancer.  Therefore, down regulation of these miRNAs at transcriptional level can reduce the probability of metastasis. This study analyzes targeting of miRNAs precursors using CRISPR-C2c2 (Cas13a) technique.

To conduct this study, we used bioinformatics and structural bioinformatics methods. The structure of C2c2 (Cas13a) enzyme was obtained from RCSB database, and the sequences of miRNAs and their precursors were collected from MirBase and Mirnamap. The crRNAs were designed, evaluated and checked for their specificity by using CRISPR-RT. The modeling of the three-dimensional structure of the designed crRNAs was performed by RNAbuilder 2.8.2 software. We used Hdock server to perform molecular docking to assess the energy level and the position of docked molecules. 

The crRNA designed for mir-126 showed high structural similarity with the situation observed in nature and appropriate orientation was obtained. In the case of crRNA designed to target mir-10b, despite high specificity, the correct orientation was not established.

Sequence-based evaluation of crRNAs designed for RNA-level editing is insufficient, and it is recommended that, along with specificity, simulation and molecular docking studies, be performed for higher accuracy.