CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Safely and effectively delivering Cas9 elements into cells is critical for gene editing based therapies. The protocols we show here display a novel approach in that direction. The protocol utilizes a new electroporation method called tube electroporation.

This leads to high cellular survival rates as well as gene editing rates. The tube electroporation method is virus, drug, and the reporter enrichment free. These are preferred in clinical applications, so with gene editing based therapies.

The method is universally applicable to many cell types. For example human hematopoietic cells, primary T cells, and other cells can all be edited using our method. This tube electroporation method uses a thin tube.

If you're a first time user, you can try GFP expressing plasmid DNA. Demonstrating the procedure will be Linyuan Ma, a postdoc from my laboratory. To begin this procedure acquire human iPSCs from the American Type Culture Collection.

Culture the iPSCs on artificial extracellular matrix with feeder-free cell culture medium in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide following the supplier's instructions. Two hours prior to transfection treat the iPSCs with 10 micromolar Rho-associated coiled-coil containing protein kinase inhibitor Y-27632. When transfecting, dissociate iPSCs with cell detachment solution to single cells at 37 degrees Celsius for five minutes.

Then count the number of cells. To establish a rabbit fibroblast cell culture obtain an ear skin biopsy from the tip of a rabbit ear. Rinse the tissue twice with DPBS containing 5%penicillin-streptomycin.

Transfer the rinsed ear tissue to a new six centimeter tissue culture dish and cut the tissue into small pieces, each about one by one millimeter. Add a few drops of FBS to prevent the tissue from drying out. After this, spread the shredded tissue in a 10 centimeter tissue culture dish and add 10 milliliters of culture medium.

Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for three to five days. Three to five days after plating use Trypsin-EDTA to digest the cells at 37 degrees Celsius for two minutes, then count the number of cells. After designing and synthesizing the gRNAs and donor oligos, prepare the cells as previously described.

Resuspend 20 to 30, 000 cells in 20 microliters of electroporation buffer. Pipette up and down carefully to produce a single cell suspension. For Cas9 RNP transfection, pre-mix two micrograms of Cas9 NLS protein with 0.67 micrograms of gRNA at room temperature for 10 to 15 minutes.

Gently mix the formed RNP complex with two micrograms of ssODN with cells. Using universal pipette tips from the electroporation kit, transfer the cell mixture to a 20 microliter electroporation tube. Place the electroporation tube into the slot of the electroporator and press go to finish.

A successful electroporation cycle is indicated by the pulse report on the display screen of the electroporator. After the electroporation transfer the human iPS cells to one milliliter of pre-warmed Y-27632 containing culture medium as previously described. Then plate the resuspended cells in one well of a 12-well cell culture plate.

Continue culturing the plate, making sure to change the culture medium every day. Y-27632 is removed from the human iPSC culture medium 24 hours post-electroporation. 72 hours after electroporation, harvest the cells.

For a rabbit fibroblast cells, use Trypsin-EDTA to digest the cells from the culture plate. For human iPSCs use cell detachment solution to digest the cells from the culture plates. Briefly centrifuge the samples at 1000 rpm for five minutes, and resuspend the cells with 350 milliliters of lysis buffer.

Incubate overnight at 55 degrees Celsius. The next day extract the genomic DNA with phenol-chloroform using standard procedures. Amplify 100 to 200 bases pairs of DNA fragments from gels using either a gel extraction kit or directly from PCR products using a PCR SV mini kit.

To determine the gene editing efficiency by bacterial colony sequencing, use a TOPO-TA cloning kit to ligate the purified PCR products into a PCR4-TOPO vector. Randomly pick up bacterial clones and sequence the inserts using a universal sequencing primer provided by the TOPO-TA cloning kit. To determine the gene editing efficiency by deep sequencing, send the previously obtained purified PCR products for CRISPR amplicon sequencing in a DNA sequencing core.

In this study a tube electroporation method is effectively used to deliver CRISPR/Cas9 RNP and ssODNs to rabbit and human cells, leading to robust, precise gene editing. First, C231Y and Q235X mutations are produced in the IL2RG gene, and the R150G mutation is produced in the SPR gene in rabbit fibroblast cells. The specific sgRNA designs and the gene editing rates determined by bacterial TA cloning are shown here.

The IL2RG C231 locus, out of the 28 clones sequenced, one carries the precise C231Y mutation, four carry insertion or deletion mutations, and the remaining 23 are wild-type. At the IL2RG Q235 locus, out of the 27 clones sequenced, two carry the precise Q235X mutation, three carry an indel mutation, and the remaining are wild-type. At the SPG R150 locus, of the 20 clones sequenced, five carry the precise R150 gene mutation, 10 carry indel mutations, and the remaining are wild-type.

Tube electroporation is then used to deliver Cas9 RNP and ssODNs to human iPSCs and target clinically relevant loci in EGFR, Mybpc3, and HBB genes. At the EGFR locus, 15.68%of alleles carry the precise point mutations, 22.75%carry indel mutations, and the remaining 61.57%are wild-type. At the Mybpc3 locus, 37.92%carry the precise four base pair TGAA deletion, 2.24%carry indel mutations, and the remaining 59.84%are wild type.

At the HBB locus, 11.5%carry the precise E6V mutation, 35.4%carry indel mutations, and the remaining 65%are wild-type. Cas9 RNP is the preferred form of Cas9 due to its smaller size in comparison to plasmid DNA. The tube electroporation is an effective method to deliver Cas9 RNP.

We are particularly interested in utilizing tube electroporation of Cas9 RNP in primary T cells for CAR T applications.