This research aims to design and deliver epigenomic editors into human cells. We do this by fusing chromatin effectors to dCas9 for targeted gene modulation without introducing toxic DNA breaks. Challenges revolve around efficiently delivering epigenome editors into cells and ensuring precise target specificity.
Additionally, achieving stable or transient gene repression remains difficult. This protocol avoids any cleaving mechanisms, reducing toxicity and genomic instability. It describes two delivery methods for controlled gene expression modulation without permanent DNA sequence alterations.
These methods can be applied to test different fusions to dCas9 and to study basic chromatin biology. Current epigenome editors can be used for genome-wide screens and have the potential for therapeutic applications. To begin, maintain K-562 cells in a flask with RPMI media supplemented with 10%FBS and penicillin streptomycin glutamine.
On the day of nucleofection, thaw CRISPRoff mRNA in a sterile RNAse-free microcentrifuge tube and gently vortex the tube. Use two micrograms of mRNA per 2.0 times 10 to the power of five cells in each well of a PCR strip tube placed on ice. Prepare the nucleofection solution according to the manufacturer's instructions and allow it to warm to room temperature for 15 minutes before nucleofection.
Harvest and count K-562 cells using an automated cell counter with trypan blue live/dead staining. For nucleofection in a strip cuvette, aliquot approximately two times 10 to the power of five cells per sample into a sterile microcentrifuge tube. Centrifuge the cells at 500 G for five minutes at room temperature.
Discard the supernatant. Add room temperature PBS to the cell pellet and centrifuge again at 500 G for five minutes. Discard the supernatant.
Resuspend the cells in the appropriate amount of nucleofector solution. Add the calculated volume of cell solution to two micrograms of CRISPRoff solution. Transfer the cell and mRNA solution to a cuvette, ensuring that bubbles do not form, as this may compromise the nucleofection efficiency.
Gently tap the Nucleocuvette to settle the cells at the bottom. Next, nucleofect the cells using the 4D-Nucleofector system with the appropriate pulse code. The pulse code may need to be optimized for different cell types.
Consult with the manufacturer if additional optimization is needed. After nucleofection, add 80 microliters of RPMI media to each used well of the Nucleocuvette. Transfer the cell suspension into a well of a 24-well plate containing 400 microliters of pre-warmed RPMI media.
Load all flow cytometry standard, or FSC files, into FlowJo by dragging and dropping them into a new worksheet. Click on all samples to begin making gates. Open a control sample by double clicking on the corresponding file.
Create a gate for live cells in an FSC-A versus SSC-A plot using the polygon tool. A live cells tab should now appear below the sample name in the list of all the samples. Right click on this gate and select copy analysis to group to apply this gate to all samples.
Double click on the live cells gate to select only live cells. Change the axes to FSC-H versus FSC-A. Draw a polygon to gate for only single cells.
To gate for guide expressing cells, double click on the single cell gate to select only single cells. Change the axes to FSC-A versus PE-CF594-A. Draw a gate for guide expressing cells using the polygon tool.
Double click on the last setup gate, either single cells or guide expressing cells. If performing a plasmid transfection where the epigenome editor encodes for a blue fluorescent protein, or BFP fusion, create a gate for BFP positive cells for day two samples. Create a gate for reporter expression by plotting BB515-A against FSC-A to identify and gate for GFP negative cells.
Then, apply this gate to all samples. After completing the gating setup, click on table editor in the upper panel. Drag populations for analysis, such as BFP positive and GFP negative cells.
In the table editor panel, click create table and copy the data before pasting it into a spreadsheet for normalization calculations. Now, subtract the number of reporter negative cells, such as GFP negative, in the control sample from all other samples. This corrects for background silencing of the reporter.
If performing a transfection, divide each value by the percentage of BFP positive cells measured on day two to normalize all values to the percentage of BFP positive cells, then multiply by 100. This yields the transfection efficiency normalized value of the edited cells. Plot the data to create line graphs that represent changes across the epigenome editing time course.
For all epigenome editing experiments, a non-targeting guide control is recommended to confirm that changes at the target loci result from epigenome editing rather than overexpression or nonspecific binding of the epigenome editor. It is important to consider the delivery method of the epigenome editor. Plasmid DNA transfection or mRNA nucleofection may be preferred given the experimental context.
Consideration of cell type, experimental timeline, delivery readout, and delivery efficacy may impact the preferred delivery method. Cells expressing high levels of BFP at two days post-transfection indicate successful transfection with the epigenome editor. Both CRISPRoff and CRISPRi show peak silencing of the gene at day five post transfection.
In mRNA nucleofection experiments, strong gene silencing is observed by day three post-nucleofection with CRISPRoff.
