Difference between revisions of "Team:Waterloo/Lab/sgRNA"
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sgRNA is a chimera of tracrRNA and crRNA, which were the original two-part biomolecule that allowed Cas9 to find and identify its target dsDNA. The purpose of fusing together the tracrRNA and crRNA was to simplify the CRISPR-Cas9 system for scientists to use more easily. Aligning with what the University of Waterloo is best known for, the lab team continued this innovation by spending part of its summer re-engineering sgRNA so that the twenty base pair target region could be removed and replaced by a new twenty base pair target region with basic classical cloning techniques. This challenge was tackled by first investigating the architecture of the sgRNA scaffold region, followed by a study of how different point mutations to the sgRNA scaffold region would affect its secondary structure. Both tasks were completed by a thorough scientific literature review that lead to the final, characterized design shown further below. | sgRNA is a chimera of tracrRNA and crRNA, which were the original two-part biomolecule that allowed Cas9 to find and identify its target dsDNA. The purpose of fusing together the tracrRNA and crRNA was to simplify the CRISPR-Cas9 system for scientists to use more easily. Aligning with what the University of Waterloo is best known for, the lab team continued this innovation by spending part of its summer re-engineering sgRNA so that the twenty base pair target region could be removed and replaced by a new twenty base pair target region with basic classical cloning techniques. This challenge was tackled by first investigating the architecture of the sgRNA scaffold region, followed by a study of how different point mutations to the sgRNA scaffold region would affect its secondary structure. Both tasks were completed by a thorough scientific literature review that lead to the final, characterized design shown further below. | ||
</p> | </p> | ||
| − | + | <figure> | |
<img src="https://static.igem.org/mediawiki/2015/d/df/Waterloo_sgRNAidea.jpg" alt="sgRNA Modification Concept"> | <img src="https://static.igem.org/mediawiki/2015/d/df/Waterloo_sgRNAidea.jpg" alt="sgRNA Modification Concept"> | ||
| − | + | <figcaption>The lower case letters represent the target sequence of the sgRNA; the upper case letters represent the scaffold region of the sgRNA. Coloured letters highlight the different modifications. The red letters highlight the newly added restriction enzyme sites, while the pink letters show the original sequence for BamHI. The lower case red is SphI, and the upper case red is BamHI. The blue letters (ie. the BamHI Complement) were modified so that they were partially complementary to the BamHI base pairs. The light blue letter show the original sequence for the BamHI Complement. </figcaption> | |
| + | <figure> | ||
<h2> The Design </h2> | <h2> The Design </h2> | ||
Revision as of 01:42, 19 September 2015
sgRNA Modification
sgRNA is a chimera of tracrRNA and crRNA, which were the original two-part biomolecule that allowed Cas9 to find and identify its target dsDNA. The purpose of fusing together the tracrRNA and crRNA was to simplify the CRISPR-Cas9 system for scientists to use more easily. Aligning with what the University of Waterloo is best known for, the lab team continued this innovation by spending part of its summer re-engineering sgRNA so that the twenty base pair target region could be removed and replaced by a new twenty base pair target region with basic classical cloning techniques. This challenge was tackled by first investigating the architecture of the sgRNA scaffold region, followed by a study of how different point mutations to the sgRNA scaffold region would affect its secondary structure. Both tasks were completed by a thorough scientific literature review that lead to the final, characterized design shown further below.
