Overview of Cas9

CRISPR is a component of the bacterial immune system against invading DNAs. When an invading DNA enters bacteria, part of its DNA is incorporated in bacterial genome and later gets transcribed as an spacer sequences in an RNA called per-crRNA. In the type II CRISPR system that we used in our study, this primary transcript in the presence of another trans encoded RNA (tracrRNA) gets processed to smaller RNA called crRNA that bares complementary sequences against invading DNA. The combinations of two RNAs bind to a protein called Cas9 which is an RNA binding DNA nuclease and guide Cas9 to the invading DNA to cleave and degrade it. In our study, we mutated Cas9 nuclease and made it catalytically inactive. It can bind to RNA and be targeted to DNA but doesn't cleave it. We also got inspired by recent publications that have used Cas9 for genetic engineering and instead of two required RNAs used one chimeric RNA that contains sequences from both crRNAs and tracrRNA called guide RNAs. The Cas9-gRNA complex then scans the DNA for a sequence complementary to the 5' nucleotides of the complexed guide RNA. Once a complementary region has been found, the Cas9 stops and binds to that location.

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.

Here is our functional 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 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 Application

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 Coculturing

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).