MODEL OVERVIEW
Our antiviral system is built around a fusion protein (gp120-DTA-Cas13a) and a set of designed guide RNAs (gRNAs) targeting a mimic HIV sequence. Within our fusion protein, the gp120 domain allows for cell-specific uptake through CD4 receptors expressed on our engineered HEK293 cells. Once the fusion protein is internalized, DTA will facilitate the entry of Cas13a into the cell. After successful entry, Cas13a forms a complex with its gRNA and will perform cleavage of viral RNA.
To better understand and optimize this system, we use computational approaches for the analysis of gRNA specificity, simulations of RNA cleavage dynamics, and analysis of our fusion protein’s stability. Our goal was to support and validate our experimental design by predicting molecular interactions and system behavior prior to wet-lab testing. Our models helped improve the design, refinement, and prediction of our engineered CRISPR-Cas13a system.
CIS- & TRANS- ACTIVITY OF CAS13A
Cis-cleavage refers to Cas13a targeting and cutting its specific target sequence, which in this case would be our HIV strand. Guided by a gRNA, the protein searches for its complementary RNA sequence. Upon finding this strand, Cas13a will bind to and cleave the target sequence. This is the intended antiviral function of our system, representing the on-target activity of Cas13a.
Once Cas13a has cleaved its target sequence, this initial action causes a conformational shift that aligns the HEPN domains into an active catalytic site. This shift activates the protein’s hyperactivation state, enabling it to indiscriminately cleave other RNAs nearby at an accelerated rate, regardless of sequence complementarity (Liu et al.).
While trans-cleavage has been researched and experimented for diagnostics, it would be a potential risk for our therapeutic design as important RNA sequences could be affected by collateral cleavage. By accounting for both cis and trans activities, we can push for our modeling framework to be more biologically realistic so that our predictions can better reflect Cas13a’s behavior in mammalian cells.
GOALS
- To prove that our mimic HIV RNA strands are able to be cleaved by the gRNA designed for our Cas13a protein
- To prove that our fusion protein (gp120-DTA-cas13a) is able to be uptaken by HEK293 cells expressing CD4 receptors
To support these goals, our strategy aims to achieve three main objectives:
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Design effective guide RNAs (gRNAs) by identifying sequences that are both specific to conserved regions of the HIV-1 genome and are in common groups within HIV-1. Analysis was performed on the sequences in order to determine the most stable gRNAs that will be used in wet lab testing.
gRNA Design Process
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Support experimental design with mathematical modeling by simulating the dynamics of Cas13a-mediated cleavage and estimating the efficiency of RNA knockdown under different conditions in MATLAB. This helps the refinement of experimental parameters and prediction of outcomes prior to validation from wet-lab results.
Computational Modeling
We also aim to determine which parameters have the greatest impact on Cas13a-mediated cleavage efficiency and model outcomes. Sensitivity analysis was conducted in MATLAB by varying key rate constants and quantifying their effect on our models. Parameters were ranked based on their influence for experimental optimization.
Parameter Sensitivity Analysis
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Utilize molecular dynamics simulations to evaluate the stability and structural integrity of our fusion protein. Generated AlphaFold structures were relaxed and simulated in explicit solvent to observe overall conformational dynamics and ensure that the Cas13a domains remain functionally intact.
Molecular Dynamics
In summary, our modeling effectively answered questions outlined in our objectives about the CRISPR/Cas13a system and provided insights into gRNA design, cleavage dynamics, and protein stability. These results laid a strong computational foundation for our experimental work, ensuring that our wet-lab designs are both informed and optimized for success.
