CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Producing genetically modified mice and analyzing their phenotype enables us to understand specific gene functions in detail, in vivo. Numerous important findings have been uncovered using gene-modified animal models. The method we describe here enables long DNA to implant stem cells with acceptable efficiency for without drug selection.

The technique is beneficial not only for basic life science, but the findings will also be implemented in applied sciences such as medical science and animal science. Demonstrating the procedure will be Dr.Chihiro Emori, an assistant professor, and Dr.Manabu Ozawa, an associate professor from my laboratory. After preparing the mouse embryonic fibroblast, prepare the gelatin-coated cell culture dish as described in the manuscript and incubate it for two hours in a humid incubator.

Remove the gelatin solution, wash the dish twice with PBS, and store it at room temperature until use. Thaw mitotically inactivated frozen MEF stock using a water bath tempered at 37 degrees Celsius for one minute. One day before ESC seeding, place the MEF on a gelatin-coated 60-millimeter dish.

Thaw a frozen ESC stock tube as shown previously and place one times 10 to the fifth ESCs onto a 60-millimeter dish containing pre-seeded MEF. Then, add four milliliters of ESC culture medium and incubate the dish until the ESC reaches 50 to 70%confluency. After washing the cultured ESC with four milliliters of PBS, digest them with 800 microliters of 0.25%trypsin EDTA solution for five minutes at 37 degrees Celsius.

Once the trypsin is inactivated with one milliliter of ESCM, add one milliliter of fresh ESCM to the cells, transfer the medium to a tube, and pellet the cells by centrifugation at 280 G for five minutes. After resuspending the cells in one milliliter of fresh ESCM, count the cell concentration using a cell counter with trypan blue staining. After transferring one times 10 to the fifth ESCs to a new 1.5-milliliter tube and washing them thrice with PBS by centrifugation, resuspend the ESC pellet in 12 microliters of Cas9 RNP DNA mixture and mix well by gentle pipetting to avoid bubbling.

Then, serve the suspended ESC for electroporation. Next, culture the electroporated ESCs in a 60-millimeter dish containing four milliliters of ECSM with mitotically inactivated MEF and keep changing the medium daily. Three to five days after the electroporation, wash the ESCs with four milliliters of PBS then digest them with 800 microliters of 0.25%trypsin EDTA and culture in a humid incubator.

Afterward, add two milliliters of ESCM to stop trypsin digestion. Once the cell mixture is pelleted, resuspend the pellet in one milliliter of fresh ESCM and count the cell concentration using a cell counter with trypan blue staining. Passage the ESCs at one times 10 to the third cells per 60 millimeter dish containing ESCM and feeder MEF.

Ensure to change the medium until the colony picks up. Five to seven days after the first passage, once the ESCM is aspirated from the ESC culture dish, add four milliliters of PBS. Using a 20-microliter pipette, pick up single ESC colonies with five microliters of PBS under a stereomicroscope.

Place the individual colonies in a well of a round-bottom 96-well plate containing 15 microliters of 0.25%trypsin EDTA solution. After incubating the 96-well plate, stop trypsin digestion by adding 80 microliters of ESCM and dissociating ESC colonies into single cells by pipetting. For PCR genotyping, transfer 40 microliters of ESC suspension to a gelatin-coated, feeder-free, 96-well plate containing 50 microliters of ESCM per well.

To make the frozen ESC stock, transfer the remaining 60 microliters of ESC suspension into a well of a gelatin-coated 24-well plate containing 500 microliters of ESCM and feeder MEF. Stock individual ESC clones cultured in the 24-well plate until they reach 60 to 80%confluency and recover individual ESC clones by trypsinization, as demonstrated earlier. After obtaining the cell pellet by centrifugation, resuspend the in 500 microliters of cell freezing medium to freeze the cells at minus 80 degrees Celsius.

For PCR genotyping, culture ESC clones as demonstrated earlier and remove ESCM from each well by aspiration before washing it with 100 microliters of PBS twice. After aspirating the PBS, add 100 microliters of lysis buffer containing proteinase K and mix well. Now transfer the ESC lysate to a new 1.5-milliliter tube and keep it in a heat chamber at 65 degrees Celsius for at least one hour.

Once the genomic DNA is dissolved in 20 microliters of DNase-free water, determine the purity and concentration of DNA using a spectrophotometer. After performing genomic PCR using locus-specific primer sets to amplify the target region, check the sequence and choose the ESC clones with the desired knock-in sequences. After mating the hormone treated females with the ICR males and collecting the oviduct, place the collected oviducts in FHM drops and recover the two-cell or four-cell stage embryos by flushing the oviduct with FHM using a flushing needle inserted into the infundibulum.

Wash the collected embryos by moving them into several fresh FHM drops using a mouth pipette. Culture the embryos in 50 microliters of KSOM drops on a 35-millimeter cell culture dish covered with mineral oil as demonstrated earlier for one day until the eight-cell or morula stage is reached. Prepare glass pipettes for holding the embryos and ESC injection using a puller and microforge.

After integrating a holding pipette containing FHM into a capillary holder connected to a microinjector, set it up on the left side of the micromanipulator. Connect an injection pipette to another microinjector and set it up on the right side. Align the two pipettes straight in the field of the microscope's view.

Make five-microliter drops of 12%polyvinyl pyrrolidone and five-microliter drops of FHM onto the same lid of a 60-millimeter dish, side by side. Cover the drops with mineral oil. Transfer the embryos in five microliters of FHM drop and add one to five microliters of the ESC suspension to the embryo-containing FHM drop.

Once the injection pipette is washed with polyvinyl pyrrolidone, move the injection pipette to the drop containing the embryos and ESCs. Pick three individual ESC doublets that have just finished their cell division in the injection pipette. Using the holding pipette, hold an eight-cell or morula stage embryo which has completed compaction by aspiration and make a hole in the zona pellucida with a piezoelectric pulse.

Expel the six-celled ESC inside the zona pellucida and pull the pipette out of the embryos. Wash the ESC-injected embryos with several KSOM drops gently using a mouth pipette and incubate them in a new 50-microliter KSOM drop covered with mineral oil, as demonstrated earlier, until the embryos developed to the blastocyst stage. A PCR amplicon of size specific to the knock-in target is obtained and 9 out of 22 clones showed a knock-in-specific band.

These results are efficient and reproducible for gene knock-in without any drug selections. Picking up the optimal size of ESC colonies is quite important for this procedure. Avoid selecting colonies that are too big or too small.

CRISPR Cas9-mediated direct genome editing using a zygote would be applicable for making simple gene knockout mice. This CRISPR Cas9-mediated ESC gene targeting protocol facilitates the production of various genetically modified mice for future analysis in life science.