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| − | Over a few weeks, Steven, Patrick, and Mark devised a method that would allow our team to swap out the gRNA segment of the sgRNA. This allowed our team to change the targets of the CRISPR/Cas9 adaptive immunity system at will. With this in mind we will first discuss the structural composition of the sgRNA scaffold, followed by a detailed explanation of the different modifications created for this "swap."
| + | 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. |
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| − | The entire sgRNA is approximately 100 base-pairs (bps) long and 20bps of this 100bps is gRNA. gRNAs are typically 20bps long due to the diminishing returns which occur with base pairs over 17 in number. By the time one reaches 20 base pairs there is no longer an advantage in creasing the number of base pairs as it has no significant effect on specificity. The other 80bps make up the scaffold which is necessary for Cas9 binding. Research shows that the gRNA length can vary among naturally occurring CRISPR arrays in bacteria and archaea, and remains full functional.
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| − | The scaffold consists of six parts. Starting at the 21st bp of the sgRNA (ie. after the gRNA), there is: the lower stem, the bulge, the upper stem, the nexus, and two hairpins. These components vary in size and number depending on the Cash. Not all bacteria use the same Cas9, meaning that there are a variety of sgRNA scaffolds corresponding to orthogonal Cas9 enzymes. Our Cas9 is derived from Streptococcus Pyogenes and will be shortened to SpyCas9 for brevity.
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| − | <h2>Briner et al. Research in sgRNA Scaffold Structure</h2>
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| − | <p>Research from Briner et al. (2014) provides an abundance of information about the effects of mutant sgRNAs on SpyCas9 functionality. To test the effects of the sgrRNA scaffold mutations, they used an in vitro and an in vivo method. The in vitro method used a biochemical assay where isolated gRNA, SpyCas9, and target DNA were mixed together in a buffered solution, followed by a diagnostic agarose gel electrophoresis experiment to determine the final lengths of DNA after this cleavage period.The in vivo method used HEK-293 cell lines that were modified to express SpyCas9 and sgRNAs. These cells were then exposed to the target DNA, followed by a T7E1 assay. One conclusion from this research was that the lower stem of the sgRNA is tolerant to individual substitutions, insertions, and deletions.
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| − | <h2>Nishimasu et al. Research into Crystal Structure</h2>
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| − | <p>Investigation of the crystal structure of the Cas9/sgRNA/DNA target structure by Nishimasu et al. (2014) identified several important features of the sgRNA/protein interface relevant to the design of the modified sgRNA. Most importantly, the base of the stem loop, referred to as the repeat-antirepeat duplex by Nishimasu et al., is a non-Watson-Crick pair, consisting of a G-U. This paper also revealed that Cas9 binds to the phosphate backbone of the helix formed by the next 5 bases, 22 to 26, and their pairs 45 to 49 of the sgRNA lower stem. This supports previous findings (Briner et al. 2014) that suggest that sgRNA binding is independent of the sequence of the lower stem. Overall, this means that the sgRNA lower stem can be replaced by any restriction enzyme site beginning with a G, and only one restriction site will be present in the DNA (see image below).</p>
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| − | <h2> sgRNA Modification</h2>
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| − | <p>The non-Watson-Crick pairing means only one restriction site is added to the DNA sequence, so only one location will be cut. This allows the scaffold RNA to be synthesized separately from the guide.
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sgRNA Modification
Introduction
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.
Experimental Design
Constructs
Target 1.0, all targets with sgRNA controls and main
Results