A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

This method provides fast, temperature controlled CRISPR-Cas9 gene editing under a small molecule, Shield-1. It also offers less off-target effects, lower cell toxicity, and also higher internally controlled gene editing when comparing it to constitutive activation of Cas9. The main advantage of this particle is that it can be used for characterization of essential genes, as well as tumor survival genes.

Destabilization of DD-Cas9 is independent of its expression, and this allows the gene of interest to be co-expressed under the same promoter. This method can be easily applied to in vivo and in vitro studies. Shield-1 can also penetrate through blood-brain barrier, which making it easy also to study genes that are involved in brain development.

Evenly seed healthy HEK293T cells at a density of one to two million cells per 10 centimeter tissue culture plate by using 10 milliliters of glucose DMEM with 10%filtered FBS. Incubate the cells in the tissue culture incubator at 5%carbon dioxide and 37 degrees Celsius for 20 hours. On the next day, confirm that the cells reached 70%confluency by brightfield microscopy and that they are evenly dispersed across the plate.

Change media one hour before the transfection with a final volume of 10 milliliters. Prepare two tubes with 500 microliters of warm media. Add 25 microliters of transfection reagent to tube one, and incubate at room temperature for five minutes.

To the other tube, add a mixture of plasmids. Mix tube one with tube two to form a transfection mixture, and incubate at room temperature for 20 minutes. Add the transfection mixture dropwise to the HEK293T cells and incubate the plate in the tissue culture incubator.

After 18 hours, carefully change the media to 10 milliliters of fresh DMEM with 10%FBS to remove the transfection reagent. Incubate the cells for the next 48 hours. After 48 hours, collect the supernatant with a 10 milliliter syringe and pass it through a 0.45 micrometer filter.

Aliquot the virus supernatant and store it at minus 80 degrees Celsius. For determining virus titer, seed 5 million HEK293T cells per well into two six-well plates. Incubate both plates at 37 degrees Celsius and 5%carbon dioxide overnight to reach 50 to 60%confluency.

On the next day, use one of the six-well plates to count the cells in the six wells. Thaw the virus aliquot that will be used to perform the serial dilution and add eight micrograms per milliliter of polybrene reagent, prepare DMEM with 10%FBS for serial dilution of the virus, and add the polybrene reagent. Prepare two milliliters of tenfold serial dilutions of the lentivirus from 0.1 to 0.0001 in polybrene containing media.

Remove media from the six-well plate and add one milliliter of viral dilutions to the wells, leaving one well with media alone as a negative control and one well with 100%virus. Incubate the cells for 24 hours. On the next day, remove the media with the virus from the six-well plates and change it to two milliliters of fresh DMEM with 10%FBS.

Incubate the cells for 48 to 72 hours, observing GFP with a fluorescent microscope every day. After 48 to 72 hours, detach the cells and resuspend them in max buffer. Use a flow cytometer to determine the percentage of GFP expression and calculate the virus titer using the formula given in the text manuscript.

Plate the cell line of interest in a 10 centimeter plate and incubate overnight to reach confluency of 50 to 60%Then infect the cells with 500 to 2, 000 microliters of virus particles in a 10 milliliter total volume of culture medium. Incubate the viral media for 24 hours. On the next day, change the viral media to culture media, and determine the percentage of GFP positive cells by using flow cytometry.

Select GFP positive cells by using BD FACSAria II for cell sorting. Expand positively selected cells and freeze the stock. Plate the positively selected cells and untransduced cells in 12-well plates separately, 24 hours after infection.

Wait until the cells attach and change the media to cell culture media containing 200 nanomolar Shield-1. Replace the media in the plates with transduced and untransduced cells, leaving two wells per plate with regular media as a negative control. Incubate and extract proteins from each well at zero, two, six, 12, 24, 48, and 72 hours after adding Shield-1 to the wells.

Then remove the media with Shield-1 from the rest of the wells and change it for regular cell media. Collect the proteins from those wells at two, six, and 12 hours after changing the media. The induction of Cas9 protein was confirmed by its expression levels being similar amongst the transduced cells and the vehicle, notwithstanding the presence or absence of Shield-1.

There is sufficient induction of Cas9 expression in the transduced A549 cell line two hours after treatment with Shield-1, which becomes negligible within six to 12 hours after withdrawal of Shield-1. Shield-1 treated transduced A549 cells decreased in number after 48 hours without affecting the Rinella sample, which was validated by immunoblotting using an antibody against the depleted RPA3 protein. Gene editing inversion or indel mutations were confirmed using surveyor nuclease assays.

The lentivirus vector construct designed was a bicistronic system bearing another gene of interest under the same EF-1a promoter. A modified fluorescent protein, P2A, was added to trace infected cells. The expression of the mVenus protein was observed in the vehicle and A549 transduced cells, independent of DD-Cas9 expression and Shield-1 treatment.

The efficient transduction of the cell line with DD-Cas9 vector is one of the rate limiting steps, for having healthy HEK293T cells and using their low passage number is crucial. The optimization of the ratio between DD-Cas9 plasmid, packaging plasmid, and envelope plasmid is very important for optimal transfection. But beside it, also the amount of Shield-1 that is needed to stabilize DD-Cas9 target cells is something that you need to be careful of.

The DD-Cas9 with the Cre plasmid is also available, and it can be used to study the genetic interactions in in vivo studies based on Cre-lox system.