This protocol uses CRISPR-Cas9 to create knock-out or knock-in lines. The main advantage of this technique is that it is simple and efficient to follow especially for novice researchers. For cell passaging, treat overnight cultured cells with two milliliters of 0.25%trypsin EDTA per plate at 37 degrees Celsius for two minutes.
When the cells have lifted from the plate bottoms, neutralize the enzymatic reaction with two milliliters of cell culture medium, and transfer the cell suspension to a conical tube. Collect the cells by centrifugation and resuspend the pellet in five milliliters of fresh cell culture medium for counting. Then seed 1.8 times 10 to the fifth cells into one well of a 24-well tissue culture plate for overnight culture at 37 degrees Celsius and 5%carbon dioxide.
For transfection of the cells, add an appropriate volume of transfection mix containing the appropriate concentration of CRISPR plasmid to the appropriate volume of transfection reagent, according to the experimental design and the manufacturer's instructions. After incubating the transfection mix at room temperature for the recommended period of time, add the solution to the cells in a drop-wise fashion. Then gently swirl the plate to mix and place the plate in a humidified 37 degree Celsius incubator with 5%carbon dioxide.
At the end of the transfection, add 150 microliters of trypsin-EDTA to dissociate the cells as just demonstrated and collect the detached cells by centrifugation. Resuspend the pellet in 2%fetal bovine serum in PBS and filter the cells through a 30 micrometer mesh strainer into a five milliliter fluorescence-activated cell sorting, or FACS tube. Then use the non-transfected cells to set a negative control gate on the flow cytometer and sort the transfected cells according to the fluorescent marker on the CRISPR plasmid used for the transfection into a tube containing 100 microliters of cell culture medium.
When all of the transfected cells have been collected, pellet the cells by centrifugation at maximum speed and resuspend the pellet in 300 microliters of cell culture medium. Seed 200 microliters of cells into one well of a 24-well tissue culture plate and allow the cells to recover for a few days in a 37 degree Celsius incubator. Pellet the remaining 100 microliters of sorted cells at maximum speed for five minutes and extract the genomic DNA from the resulting pellet according to standard DNA extraction protocols.
Next, add 10 microliters of polymerase chain reaction buffer, one microliter of deoxynucleotide mix, 2.5 microliters of user-defined reverse primer, 0.5 microliters of DNA polymerase, two to five microliters of the extracted genomic DNA template and enough double-distilled water to bring the reaction to a final volume of 50 microliters. Run the mixture on a thermal cycler and resolve the reaction on a 2%agarose gel using 1X TAE buffer according to standard protocols. Use a clean, sharp scalpel to excise the polymerase chain react product and purify the DNA using a gel extraction kit according to the manufacturer's instructions.
Measure the concentration of the product using a spectrophotometer at 260 nanometer absorbance. Prepare an assay mix containing 200 nanograms of the isolated DNA, two microliters of T7 endonuclease I reaction buffer, and enough double-distilled water to bring the final volume of the mixture to 19 microliters. Reanneal the polymerase chain reaction product in a thermal cycler at the indicated parameters and mix five units of T7 endonuclease I with the reannealed product for a 50 minute incubation at 37 degrees Celsius.
At the end of the incubation, resolve the digested DNA on a 2.5%agarose gel using 1X TAE buffer, and image the gel on an appropriate gel imaging system. Open the gel image in ImageJ and draw a rectangular box around the band as close to its boundary as possible. Click Analyze, and Set Measurements, confirming that the area, mean gray value, and integrated density options are checked.
Click OK, and select Analyze, and Measure. The mean, or raw intensity density value, is indicative of the band intensity. When the culture-transfected cells start becoming confluent, detach them with trypsin-EDTA, as demonstrated, and seed the cells sparsely in a 100 millimeter tissue culture dish to allow sufficient space for individual colonies to grow before returning the cells to the cell culture incubator.
When the colonies begin to form, use a microscope with a four X magnification to pick the individual colonies for transfer into individual wells of a 24-well plate containing 500 microliters of cell culture medium per well. Take care that your tip does not touch the surrounding colonies to prevent mixing of colonies within individual wells or pick from an area of sparse colony growth. When all of the clones have been picked, place the plate in the cell culture incubator until the cultures become confluent.
As undigested plasmids are super-coiled, they tend to run faster than their linearized counterparts. To determine if the oligonucleotides have been successfully cloned into the CRISPR plasmid backbone, colony PCR is performed and the positive clones are inoculated before the plasmids are extracted and sent for Sanger sequencing. The fluorescent signal can be readily visualized under a microscope upon a successful delivery of the plasmids, allowing the transfected cells to be sorted by flow cytometry.
A T7 endonuclease I assay is performed to check the cleavage efficiency of the genomic DNA, as calculated from the intensities of the bands observed on an agarose gel. Additionally, if a homology-directed repair-based experiment is designed to incorporate a restriction site at the target locus, a restriction fragment length polymorphism assay can be performed with a corresponding restriction enzyme. To further validate that a protein coding gene has been successfully inactivated, a Western blot can be carried out to ensure that no targeted protein is present.
Sorting the cells after transfection eliminates the cells without incorporated plasmids, thereby increasing the percentage of cells screened at later stages as having a knock-out or knock-in gene. With the development of this technique, we are now able to produce knock-out cell lines to study gene function and knock-in cell lines to model specific diseases to understand their mechanism.
