シームレスなゲノム編集によるヒト多能性幹細胞への点突然変異の導入

Editing genes in human pluripotent stem cells is challenging. This protocol provides the detail procedure for genome editing in these cells using paired Cas-90 case combined with the targeting vector. It is reliable and easy to follow.

The main advantage of this protocol is that it is seamless genomic editing. The two-step selection helps to increase targeted cells and has made the genotyping screening more efficient. Maintain human pluripotent stem cells on recombinant laminin 21 coded surfaces in mTeSR1 medium.

The cells should reach 70 to 85%confluency, 72 hours after a one to three split ratio. On the day of transfection coat enough wells of a 24 well plate with 300 microliters of the laminin 521 solution, and incubate the plate at 37 degrees Celsius for at least two hours. After the incubation gently remove the coating solution and immediately add 300 microliters of fresh mTeSR1 medium, supplemented with 10 micromolar ROCK inhibitor to each well.

Then put the plate back in the incubator. Take the stock human pluripotent stem cells out of the incubator. Remove spent medium and wash the cells once with one milliliter of sterile DPBS.

Then add one milliliter of gentle cell dissociation reagent to each well and incubate it at 37 degrees Celsius for six to eight minutes to dissociate the cells. Gently tap the plate to make sure that the cells can detach easily. Then use a P1000 tip to lift the cells by pipetting up and down.

Add two milliliters of mTeSR1 medium with ROCK inhibitor to terminate the dissociation. Mix the suspension well and transfer it to a 50 milliliter tube. Centrifuge the cells at 200 G for three minutes.

Then remove the supernatant and resuspend the cells in two milliliters of mTeSR1 medium with ROCK inhibitor. Count the cells using a hemocytometer and trypan blue. Transfer 800, 000 to 1 million cells to a 1.5 milliliter tube for each nucleofection reaction and centrifuge them at 200 G for three minutes.

Then carefully aspirate the supernatant. Mix three micrograms of the paired Cas-9n single guide RNA expression plasmids and five micrograms of targeting vector plasmid in 100 microliters of mixed nucleofection solution from the human stem cell nucleofection kit. Prepare a GFP control plasmid mix as well.

Resuspend the cells with the DNA mix and transfer them to an electroporation cuvette, making sure to avoid air bubbles. Electroporate the cells with the nucleofection device using the optimized condition for human pluripotent stem cells. Immediately add 500 microliters of fresh and warm mTeSR1 medium supplemented with 10 micromolar ROCK inhibitor to the electroporated cells.

Then transfer the mix to two wells of the previously prepared 24 well plate. Put the plate in a 37 degree Celsius incubator supplied with 5%carbon dioxide to allow the cells to recover. Replace the cell maintenance medium after 12 to 16 hours, withdrawing the ROCK inhibitor if the cells establish cell to cell contact.

After 24 to 48 hours, check the nucleofection efficiency by examining GFP expression in the control cells. Start selecting cells by supplementing the mTeSR1 medium with one microgram per milliliter puromycin, 48 hours after nucleofection. After 72 hours supplement the medium with 0.5 micrograms per milliliter puromycin.

If the cell confluency is lower than 30%also supplement the medium with 10 micromolar ROCK inhibitor. Four to six days after nucleofection passage the puromycin resistant cells to 10 to 15 96 well plates at the concentration of 0.8 cells per well. Making sure to supplement the medium with ROCK inhibitor and puromycin.

Maintain the cells at 37 degrees Celsius and 10%carbon dioxide for 10 to 12 days to form single cell derived colonies. Topping up the medium seven days after seeding. Mark wells containing a single colony and replace the medium with fresh mTeSR1 medium, containing 0.5 micrograms per milliliter puromycin, but no ROCK inhibitor.

Grow the cells for two more days at 37 degrees Celsius and 5%carbon dioxide. Then change medium for wells containing undifferentiated colonies. Gently scrape off the colonies and transfer the cell suspension from one colony into two new wells on separate 96 well plates.

One for genotyping, and one for maintaining. Once the confluency of the genotyping cells reaches 50%or above, dump the spent medium and wash the cells once with DPBS. Lyse the cells with Bradley Lysis Buffer and isolate genomic DNA from each well.

After performing three primer junction PCR and sequencing the products, keep the colonies with the correct genotype and discard the rest. Expand the correct colonies under continuous puromycin selection, and freeze them at the earliest possible passage. This protocol was used to introduce two point mutations into Exon8 of the hepatocyte nuclear factor four alpha gene.

A three primer based PCR method was used to screen for correctly targeted cells, and Sanger sequencing was performed to confirm the PCR results. After the removal of the selection cassette the modified region was sequenced again to confirm the correct introduction of desired point mutations. Colonies with the correct genotype were selected and the cells were characterized prior to further analysis.

The edited cells possess the same morphology as the parental cells and express representative human pluripotent stem cell markers including transcription factors Nanog and Oct4, as well as cell surface markers, SSEA-4 and TRA-160. When attempting this procedure it is important to remember to check the cell confluency after puromycin selection during the first 24 hours. It is essential to supplement the median with ROCK inhibitor.

If the cell confluency is lower than 30%to keep the cells going. Once the genome edited pluripotent stem cell lines have been established they can be used to study gene function or directed differentiation. This technique paves the way for researchers to study different questions, such as the specific function of genes or targeted gene correction for genetic disease.