Design

Simple sgRNA Exchange

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Cas9 PAM Flexibility

Being able to target DNA at different protospacer adjacent motif (PAM) site will allow scientists to target a larger portion of the genome. "Dead" Cas9 (dCas9) allows for easier analysis of products because the DNA still remains intact. Kleinstiver et al proposed an EQR variant of Cas9 with three amino acid substitutions. This new proposed version of Cas9 targeted an NGAG PAM sequence. Our design was to make these three amino acid substitutions in dCas9 and try to target an NGAG PAM site in GFP.

Kleinstiver et al. recently demonstrated modified spCas9 with altered PAM specificity . Their results motivated us to explore computational methods of assessing spCas9 mutants for altered PAM specificity profiles. We created an analysis pipeline using python that makes use of PyRosetta, a well known molecular dynamics toolkit. Our suite of scripts is described in our modeling and software pages.

Wild Type spCas residues — A PyMOL generated image of the wild type residues near the PAM binding site of spCas9.

EQR Mutated Residues — A PyMOL generated image of the three mutations found in the EQR spCas9 variant

VQR Mutated Residues — A PyMOL generated image of the three mutations found in the VQR spCas9 variant

CRISPR Plant Defense

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To model our antiviral application, we looked at the antiviral effects of CRISPR/Cas9 targeting on three scales: CaMV genomes, plant cells and plant leaves. Details of our modeling approach, and how it influenced our design, are provided in our Modeling pages.

References

By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page. If you are going for the Applied Design award, you should also complete this page and tell us what you did.

Note

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This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.

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