The study of gene function and disease are hampered by the outbred nature of the human population. The different genetic backgrounds of patient-specific and control iPS cell lines can affect differentiation efficiency and phenotypes found in a given functional assay. Use of genetically identical or isogenic lines where the only difference is the particular mutation of interest is critical to overcome these issues.
Many human diseases are caused by heterozygous mutations, which can be difficult to generate with the CRISPR-Cas9 system. This is due to the generation of indel mutations in the non-targeted allele that can occur at very high frequency. Our technique overcomes this problem by using two repair templates of which only one harbors the mutation of interest.
Any investigator trying this technique should be skilled at human pluripotent stem cell culture. The video demonstration of colony picking will be helpful to show correct size and morphology as well as technique used for picking and screening. Demonstrating this procedure will be Leo Cardenas.
To begin, plate human ESCs on irradiated MEFs in a 6-well plate. Prepare the transfection master mix. Mix by pipetting and incubate at room temperature for 15 minutes.
When the cells reach 70 to 80%confluency, then, add the transfection master mix drop wise to each well with cells and incubate at 37 degrees Celsius for 48 hours. Change the media every 24 hours. To harvest the cells, first, incubate the cells with Triple E for three minutes at room temperature to remove MEFs enzymatically.
Add 0.5 milliliters of HESC medium with 10 micromolar ROCK inhibitor. Pellet cells at 300 times g for three minutes and re-suspend in 0.5 milliliters of human ESC medium with 10 micromolar ROCK inhibitor. Filter the cell suspension into a five milliliter tube through a 35 micron cell strainer cap.
Using fluorescence-activated cell sorting, gate on live cells and sort the green fluorescent protein-positive cells. Next, transfer a maximum of 1.5 times 10 to the four sorted cells directly into a 10 centimeter dish coated with one to three basement membrane matrix and irradiated MEFs and human ESC medium containing ROCK inhibitor. Change medium daily using the human ESC medium without ROCK inhibitor.
After 10 to 15 days, colonies are approximately one to two millimeters in diameter. Under a microscope, use a P200 pipette to carefully scrape a single clone and draw cells into the pipette. Disperse the cells in a well of a 96-well plate by gently pipetting three to four times in the medium drawn up with the colony.
Then, pick 20 colonies for each guide RNA and dispense into PCR strip tubes for screening. Pellet the cells by centrifugation at 10, 000 times G for five minutes. Now, incubate cell pellets in 20 microliters of Proteinase K buffer to isolate DNA and vortex vigorously.
Centrifuge at 10, 000 times G for five minutes. Next, perform screening PCR of the edited DNA. Add into the tubes 20 microliters of a master mix, including forward and reverse primers, designed to amplify the region of interest, as well as five microliters of the Proteinase K digest.
Use genomic DNA isolated from the control iPSC line to confirm a clean amplicon when performing the screening PCR. Evaluate size changes of PCR products following one hour of electrophoresis at 70 to 90 volts on a 2.5%weight by volume agarose gel. Any size difference is indicative of cleavage.
To transfect the single strand oligo DNA and the CRISPR-Cas9 plasmids, plate the target cell line in a 6-well dish on irradiated MEFs to reach 70 to 80%confluency after over an overnight incubation. Set up the transfection reaction as described. Mix the reaction by pipetting and incubate for 15 minutes at room temperature.
Then, add the transfection reaction mixture drop wise to the cells. After 48 hours, prepare the cells for cell sorting as previously. About 10 days after plating single cells, use a 200 microliter pipette to pick colonies into PCR strip tubes and into a well of a 12-well plate pre-coated with gelatine and MEFs.
Transfer 100 microliters of the cells to each well of a 24 or 48-well plate, previously coated with gelatine and irradiated MEFs in human ESC medium with ROCK inhibitor. Use the remaining 100 microliters for DNA isolation. To check for successful integration of the single strand oligo DNA, take five microliters of DNA isolated from each colony to perform PCR using the screening primers previously designed.
Purify the PCR products and prepare the 40 microliters of restriction enzyme digestion using the unique enzyme site created in the single strand oligo DNA. Mix the reaction by pipetting and incubate at the manufacturer's recommended temperature for one to three hours. Then, visualize the digested PCR products added with a loading dye on a 1.5%weight by volume ethidium bromide agarose gel, electrophorese at 80 to 100 volts for 40 minutes.
If successful integration of the single strand oligo DNA has occurred, sequence specific mutations using a nested primer. In this study, for guide RNA construction, a 100 base pair band was excised from a 1.5%agarose gel. Following cell transfection, validation of guide RNA cutting was visualized using a 2.5%agarose gel.
A 180 base pair PCR product showed an uncut control and different clones with band shifts indicating indel formation. Different guide RNAs had different cutting efficiencies. To avoid CRISPR-Cas9 re-cutting of the edited alleles, the PAM sequence was modified using a G to A silent mutation.
A silent mutation in the PAM sequence generated a unique restriction site, which helps to screen for successful integration into one or two alleles. Genome edited stem cell lines can be used in downstream differentiations and functional assays to study the effect of a given mutation on human development and disease. This methodology allows the generation of isogenic cell lines with a specific heterozygous or homozygous mutations, implicated in a wide variety of human diseases.
These disease models pave the way for the development of new cellular therapies as well as offer platforms for drug discovery.
