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Team:MIT/Cas9-VP16
From 2013.igem.org
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The following data was taken using the Life Technologies EVOS fluorescent microscope 48 hours post transfection. The results of the graph above are recapitulated in the increase in visible fluorescence as transfected guide RNA amount increases. Click on the images to open up in a separate window. | The following data was taken using the Life Technologies EVOS fluorescent microscope 48 hours post transfection. The results of the graph above are recapitulated in the increase in visible fluorescence as transfected guide RNA amount increases. Click on the images to open up in a separate window. | ||
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<h1>Exosome Isolation and Co-culturing</h1> | <h1>Exosome Isolation and Co-culturing</h1> | ||
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<h1>Cell-Cell Co-culturing</h1> | <h1>Cell-Cell Co-culturing</h1> | ||
Revision as of 04:35, 27 September 2013
Overview of Cas9
Cas9 is one of the proteins involved in the adaptive immune system (CRISPR system) of some bacteria and most archaea. In simple terms, the Cas9 functions by binding to a small hairpin RNA called a guide RNA. The Cas9-gRNA complex then scans the DNA for a sequence complementary to the 5' amino acids of the complexed guide RNA. Once a complementary region has been found, the Cas9 cuts the DNA at that location.
The Cas9 we use has been mutated such that it still complexes with the guide RNA and targets the sequence complementary to the guide RNA, but the Cas9's nuclease activity has been eliminated. Once the Cas9 finds its target site, it simply stays put.
Cas9-VP16 Characterization
Here, we describe the creation and testing of a Cas9-VP16 fusion protein. Current transcriptional level activators/repressors function by binding DNA at certain conserved sequences and influencing the transcriptional abilities of some nearby promoter. The issue with these current activators/repressors is that each one binds one unique DNA sequence. The GAL protein binds UAS sites, TetR binds TetO sites, LacI binds LacO sites, and so on. By using Cas9 as the DNA binding portion of a trans-activator, we can eliminate the need for specific binding sites by taking advantage of Cas9's unique ability to target most any DNA sequence as determined by its complexed guide RNA. The technology could be used to either target and activate endogenous sequences by creating a guide RNA which targets a sequence upstream of the promoter in question, or one could create many different guide RNAs which target different inducible promoters and activate multiple genes with one single trans-activator. Our project looks into creating a Cas9-VP16 protein and testing its ability to activate a minimal CMV promoter by targeting the fusion to two upstream binding sites using a complementary guide RNA.
The Circuit
The U6_gRNA(Cr9) produces guide RNAs to target Cas9-VP16 to the Cr9 binding sites upstream of the minimal CMV promoter. When the Cas9-VP16 activator binds to the Cr9 sites, the minimal CMV promoter can be activated and eYFP is produced. TagBFP functions as our transfection marker.
The Data
The experimental procedure involved cotransfecting the genetic circuit shown above while varying the amount of transfected guide RNA (and using a dummy construct to keep a constant transfected DNA amount) to determine when we achieve optimal activation. The circuit was transfected into 200,000 HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. The following set of data was gathered 72 hours post transfection via flow cytometry. The data is presented as median fluorescence values for subpopulations of cells grouped by intensity of transfection marker (normalizing fluorescence via plasmid count).
On the Y axis, we see the output fluorescence in the FITC channel and our transfection marker fluorescence in the Pacific A channel on the X axis. As expected, we see activation when all components of the system are present, and increasing the amount of transfected guide RNA increased the ability of the Cas9-VP16 to bind to the synthetic upstream Cr9 regulatory sites of the reporter construct. The graph indicates that saturation hasn't occured, but due to limitations of the Lipofectamine transfection protocol, transfecting the amount needed for saturation would likely be toxic to the cells.
The following data was taken using the Life Technologies EVOS fluorescent microscope 48 hours post transfection. The results of the graph above are recapitulated in the increase in visible fluorescence as transfected guide RNA amount increases. Click on the images to open up in a separate window.
Exosome Isolation and Co-culturing
These experiments will entail the creation of an Acyl-TyA-Cas9-VP16 fusion protein which could be targeted into exosomes. The isolated exosomes would then be applied to HEK293 receiver cells transfected with the reporter contruct and Cr9 targeting guide RNA and assayed for activation. We would expect that cells treated with exosomes would absorb the exosomes and release Acyl-TyA-Cas9-VP16 into the receiver cells to induce activation of the reporter construct.
Cell-Cell Co-culturing
Finally, if the exosome treatments function (indicating that our Acyl-TyA-Cas9-VP16 is being taken up by receiver cells in functional quantities) then the we'll use Jurkat T cells transfected with constitutively active Acyl-TyA-Cas9-VP16 and placed in the same well with HEK293 receiver cells transfected with the reporter and Cr9 targeting guide RNA. We'd expect the Jurkat T cells to produce Acyl-TyA-Cas9-VP16, target it into exosomes, and excrete the exosomes into the media. The receiver cells would then absorb the signal containing exosomes and react accordingly (activation of the reporter construct).
