Difference between revisions of "Team:BostonU/App 2/Results"

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<h3>Results</h3>
 
<h3>Results</h3>
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<p>We were so excited to have researched SaCas9 and found potentially viable split sites. We were able to create 16 different split sites within the sequence and cloned most of these constructs into the FKBP/FRB dimerizable domains. Utilizing transient transfection we tested the full SaCas9 and traffic light reporter however there was neither GFP nor mCherry expressed, so we concluded that no double strand break had been made. We hypothesize that there was likely an issue with our choice of target sequence within the traffic light reporter. Below we have results from our flow cytometry run showing arbitrary units of fluorescence.<p>
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<p>We were excited to have identified 16 candidate split sites, and cloned most of these split saCas9 constructs into the FKBP-FRB dimerizable domain backbones, in both orientations. We set up our flow cytometry experiments similarly to our integrase and RDF ones, except used our traffic light reporter and new guide RNA designs.
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When performing flow cytometry, we measured mCherry (NHEJ) and GFP (HDR) fluorescence values in arbitrary units. Each transfection condition was performed in triplicate on every plate, and thus we were able to obtain the mean and standard deviation across three replicates. However, the results that we obtained using flow cytometry were not optimal. Below is a result of our conditionally dimerizable saCas9 variants (in both domain orientations). <p>
  
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<center><img style="height:50%; width:50%;" src="https://static.igem.org/mediawiki/2015/thumb/a/aa/Sacas9_gfp_finalized.png/800px-Sacas9_gfp_finalized.png" />
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<center><img style="height:50%; width:40%;" src="https://static.igem.org/mediawiki/2015/thumb/d/dd/Sacas9_gfp_finalized_.png/800px-Sacas9_gfp_finalized_.png" />
 
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<img style= "height:50%; width:50%;" src="https://static.igem.org/mediawiki/2015/thumb/2/2d/Sacas9_mcherry_finalized.png/800px-Sacas9_mcherry_finalized.png" />
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<img style= "height:50%; width:40%;" src="https://static.igem.org/mediawiki/2015/thumb/2/2d/Sacas9_mcherry_finalized.png/800px-Sacas9_mcherry_finalized.png" />
 
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<p>It is clear that even the fully functional SaCas9 did not work properly, therefore none of the subsequent split SaCas9 worked either. Although we were not able to draw any conclusions from this data in the future we would like to debug our initial SaCas9 design and then continue to validate splits in the same workflow as the integrases where we will look for patterns between split site locations, different dimerization domains, and their orientations. It would be very exciting to find viable split sites and create a system for genomic editing using split SaCas9.</p>
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<p>We noticed that we got a low raw fluorescence signal using this reporting system, even with the intact saCas9. Because of this, we did not calculate or plot normalized % GFP or % mCherry for this system. We believe that our negative results are likely due to our modified gRNAs using the traffic light reporter system - these might not have optimally functioned, thus resulting in minimal saCas9 activity. We would debug our initial experimental workflow by adding in an additional control - characterizing how spCas9 (for which the traffic light reporter system was initially designed) functioned. If we had a functional reporter system, we could properly characterize our split saCas9 variants and identify any viable splits and interesting trends.
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Despite unsuccessful flow cytometry results, we had another interesting finding to share. Towards the end of our project (at the very end of August), the Feng Zhang group at MIT published the crystal structure of saCas9 in the journal Cell7. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we originally wanted to accomplish.  
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One facet worth noting is that the Zhang group used the 3D structure of the saCas9 protein to target flexible linker regions that would serve as potential split sites. Out of the three split sites that they chose, the one that worked best was almost identical (one amino acid away) to one of the split sites that we identified using our model. Furthermore, another split sites that they chose happened to be fairly close to one that we chose. Finally, the third split that they created was intended to be a negative control - we did not pick any split sites near this split, and it happened to be located in a region with secondary structural elements.
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This important result gave us some experimental validation that our model could offer scientists insights into choosing promising viable split sites for proteins, without only relying on 3D structures of proteins. We also predict that our two split saCas9 constructs chosen near the Zhang lab sites would likely be viable, though we would need to experimentally confirm this.
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</p>
  
 
<p>Towards the end of our project, the Feng Zhang group at MIT published the crystal structure of saCas9 in Cell<sup>1</sup>. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we wanted to accomplish. </p>
 
<p>Towards the end of our project, the Feng Zhang group at MIT published the crystal structure of saCas9 in Cell<sup>1</sup>. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we wanted to accomplish. </p>

Revision as of 23:00, 18 September 2015

Motivation Design Results

Results

We were excited to have identified 16 candidate split sites, and cloned most of these split saCas9 constructs into the FKBP-FRB dimerizable domain backbones, in both orientations. We set up our flow cytometry experiments similarly to our integrase and RDF ones, except used our traffic light reporter and new guide RNA designs. When performing flow cytometry, we measured mCherry (NHEJ) and GFP (HDR) fluorescence values in arbitrary units. Each transfection condition was performed in triplicate on every plate, and thus we were able to obtain the mean and standard deviation across three replicates. However, the results that we obtained using flow cytometry were not optimal. Below is a result of our conditionally dimerizable saCas9 variants (in both domain orientations).

               

We noticed that we got a low raw fluorescence signal using this reporting system, even with the intact saCas9. Because of this, we did not calculate or plot normalized % GFP or % mCherry for this system. We believe that our negative results are likely due to our modified gRNAs using the traffic light reporter system - these might not have optimally functioned, thus resulting in minimal saCas9 activity. We would debug our initial experimental workflow by adding in an additional control - characterizing how spCas9 (for which the traffic light reporter system was initially designed) functioned. If we had a functional reporter system, we could properly characterize our split saCas9 variants and identify any viable splits and interesting trends. Despite unsuccessful flow cytometry results, we had another interesting finding to share. Towards the end of our project (at the very end of August), the Feng Zhang group at MIT published the crystal structure of saCas9 in the journal Cell7. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we originally wanted to accomplish. One facet worth noting is that the Zhang group used the 3D structure of the saCas9 protein to target flexible linker regions that would serve as potential split sites. Out of the three split sites that they chose, the one that worked best was almost identical (one amino acid away) to one of the split sites that we identified using our model. Furthermore, another split sites that they chose happened to be fairly close to one that we chose. Finally, the third split that they created was intended to be a negative control - we did not pick any split sites near this split, and it happened to be located in a region with secondary structural elements. This important result gave us some experimental validation that our model could offer scientists insights into choosing promising viable split sites for proteins, without only relying on 3D structures of proteins. We also predict that our two split saCas9 constructs chosen near the Zhang lab sites would likely be viable, though we would need to experimentally confirm this.

Towards the end of our project, the Feng Zhang group at MIT published the crystal structure of saCas9 in Cell1. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we wanted to accomplish.

One facet worth noting is that the Zhang group used the 3D structure of the saCas9 protein to target flexible linker regions that would serve as potential split sites. Out of the three split sites that they chose, the one that worked best was almost identical (one amino acid away) to one of the split sites that we identified using our model. Furthermore, the other two split sites that they chose which were not as successful happened to be located in regions with secondary structural elements.

This important result gave us some experimental validation that our model could offer scientists insights into choosing promising viable split sites for proteins, without only relying on 3D structures of proteins.

Citations

  1. Nureki, Osamu et al., “Crystal structure of staphylococcus aureus Cas9”, Cell, 2015.