Overview

Model Overview

Our CRISPR/Cas13a system primarily consists of a specific Cas protein, LwaCas13a, and a designed crRNA that targets miR-21-5p. The formation of LwaCas13a/crRNA/miR-21-5p ternary complex activates its cleavage activity, which cleaves reporter RNA to release fluorescence. Therefore, the ternary complex can be treated as an enzyme, and the specificity, stability and efficiency of this enzyme can be analyzed with a variety of mathematic models. Specifically, our modeling mainly revolves the following four questions:

  • Question One: Which miRNA to select?
  • Question Two: What type of crRNA to choose?
  • Question Three: Is the assembled ternary complex stable?
  • Question Four: Is this ternary complex efficient as an enzyme (i.e., does the proposition hold that higher miRNA concentration leads to higher enzymatic activity, resulting in a faster reaction rate and ultimately higher fluorescence intensity)?

To answer Question One, we conducted bioinformatics analysis on existing databases (TCGA-BRCA, GSE59247), which primarily included differential expression analysis, feature importance analysis based on random forests and SHAP values, survival analysis, and literature validation, and identified miR-21-5p as the optimal biomarker for our Brefast detection.

Bioinformatics Analysis

To address Questions Two and Three, we designed four different crRNA sequences based on the sequence of miR-21-5p: crRNA1, crRNA2, crRNA3 and crRNA full length. Firstly, the thermodynamic characteristics of these crRNAs were analyzed by RNAfold of ViennaRNA using three criteria: minimum free energy of crRNA and miRNA complex, minimum free energy of crRNA, and GC content. Secondly, the interactions between crRNA and LwaCas13a protein were investigated by molecular docking. Lastly, molecular dynamics simulation was performed to verify the stability of the ternary complex. In the end, crRNA full length was confirmed as the best crRNA to form the CRISPR/crRNA system for interaction with miR-21-5p.

Simulation and Verification of CRISPR-Cas13a System

To answer Question Four, we simulated the cleavage reaction mediated by the CRISPR/Cas13a system through establishing a reaction kinetics equation, and validated it with experimental data, which confirmed that our experimental data indeed adhered to the reaction kinetics equation, thus substantiating the proposition in Question Four.

Reaction Kinetics Simulation

In summary, our modeling answered four important questions about the CRISPR/Cas13a system effectively, and paved the way for the subsequent web lab work.