CRISPRコンカテマーを用いたマウス小腸オルガノイドにおける複数の遺伝子ノックアウトのためのプロトコル

The overall goal of this protocol, is to clone multiple guide RNAs into one CRISPR-concatemer vector and to achieve highly efficient electroporation in mouse intestinal organoids, in order to obtain simultaneous knockout of multiple genes. This method can greatly improve the efficiency of lost of function studies by making it possible to overcome the potential effect of parallel compensation. The main advantage of our CRISPR-concatemer strategy are the convenience of a single cloning step and the possibility of performing simultaneous knockout of up to four different genes.

Demonstrating the procedure will be done by, Alessandra Merenda, a PhD student from my laboratory. The cloning of guide RNAs or gRNAs into the CRISPR-concatemer vector begins with a single reaction to anneal the top and bottom strands for each gRNA oligonucleotide and to phosphorylate their ends. Prepare the reaction mixture on ice, for three concatemers, use three microliters of top strand gRNAs, three microliters of bottom strand gRNAs, two microliters of T4 DNA ligase buffer, one microliter of T4 polynucleotide kinase and water up to a total volume of 20 microliters.

Mix well by pipetting and running the thermocycler using the following settings, 37 degrees celsius for 30 minutes, 95 degrees celsius for five minutes, ramp down to 25 degrees celsius at 0.3 degrees celsius per minute and hold at four degrees celsius. The next step is the Bbs1 shuffling reaction, in which the pre-annealed gRNA oligonucleotides are incorporated into appropriate position of the concatemer vector. Dilute the reaction mixture one to 100 in DNA, RNA free water to generate three and four gRNA concatemer vectors.

Assemble the Bbs1 shuffling reactions on ice. Use 100 nanograms of CRISPR-concatemer vector, 10 microliters of oligo mixture, one microliter of restriction enzyme buffer, one microliter of DTT, one microliter of ATP, one microliter Bbs1 enzyme, one micrliter of T7 ligase and water up to a total volume of 20 microliters. Mix well by pipetting, include a negative control that contains the vector.

Run the reactions in a thermocycler, using the following settings, for cloning three and four gRNA concatemers, run 50 cycles at 37 degree celsius for five minutes and 21 degrees celsius for five minutes, hold at 37 degree celsius for 15 minutes and then hold at four degree celsius indefinitely. For two gRNA concatemers, run the same settings with 25 cycles of step one. When the Bbs1 shuffling reaction is complete, take 11 microliters of each ligation mix and add 1.5 microliters of exonuclease buffer, 1.5 microliters of ATP, one microliter of DNA exonuclease and water to a total volume of 15 microliters.

Incubate at 37 degree celsius for 30 minutes followed by 70 degree celsius for 30 minutes. Subsequently, the reaction mixture is used to transform chemically competent E.Coli bacteria, following a standard protocol. To confirm the presence of gRNA inserts in the CRISPR-concatemer vector, pick bacteria colonies with an inoculating loop and inoculate each in four milliliters of LB medium.

Grow the clones overnight at 37 degrees celsius in an orbital shaker. On the following day, DNA is extracted using a plasmid miniprep kit, according to the manufacturer's instructions. Mix approximately 200 nanograms of each DNA sample with 10 units of EcoR1 and five units of BglII in a 10 microliter reaction.

Include a separate reaction mixture with the corresponding original vector as a positive control for size comparison. Incubate the reactions at 37 degrees celsius for three hours. Run the digestion reactions on a 1%agarose gel at 90 volts for approximately 20 minutes.

Visualize the gel using a UV transilluminator to identify the clones with the correct insert size. To confirm that gRNAs have been cloned into the right position and consequently all Bbs1 recognition sites have been lost, digest the selected clones with five units of Bbs1. Include a separate reaction mix, with the corresponding original vector as a control.

Incubate at 37 degrees celsius for three hours, run the digestion reactions on a 1%agarose gel at 90 volts for approximately 20 minutes. Visualize the gel using a UV transilluminator to identify the vectors that are not cut by Bbs1. When splitting organoids for electroporation, seed a minimum of six wells of a 48 well plate per transvection.

Seed the organoids in 20 microliter basement matrix drops and grow them in 250 microliters of WENR plus nicotinamide medium per well at 37 degrees celsius and 5%carbon dioxide in a humidified incubator. On day two, replace the WENR plus nicotinamide medium with 250 microliters of EGF plus noggin, GSK3 inhibitor and rock inhibitor medium without antibiotics. On day three, change the organoid medium to EGF plus noggin, GSK3 inhibitor, rock inhibitor and 1.25%dimethyl sulfoxide without antibiotics.

On day four, disrupt the basement matrix domes, using a one milliliter pipette tip and transfer organoids to a 1.5 milliliter tube. Pull the contents of four wells of a 48 well plate into one tube. Mechanically break the organoids into small fragments by pipetting up and down with the P200 pipette approximately 200 times.

Centrifuge at 600 times G for five minutes at room temperature. After centrifugation, remove the medium from all tubes and re-suspend the palettes in one milliliter of a cell culture grade recombinant protease. Incubate at 37 degree celsius for a maximum of five minutes.

It is important to carefully monitor the protease treatment because the success of the electroporation, depends on obtaining a cell suspension that contains clusters of cells instead of single cells. Check a 50 microliter drop of sample under an inverted light microscope with a four times objective. Clusters of 10 to 15 cells are desirable as this increases cell survival after electroporation.

Transfer the cell suspension to a low binding 15 milliliter tube and hog the dissociation by adding nine milliliters of basal medium without antibiotics. Centrifuge it at 600 times G for five minutes at room temperature. Discard the supernatant and re-suspend the palette in one milliliter of reduced serum medium.

Count the number of cells with a burkes chamber, a minimum of one times 10 to the fifth cells is needed per electroporation reaction. Begin this procedure by adding nine milliliters of reduced serum medium to the 15 milliliter tube containing the cell suspension and centrifuge at 400 times G for three minutes at room temperature. Remove all the supernatant and re-suspend the palette in an electroporation solution.

Add a total amount of 10 micrograms DNA to two new tubes. Then, add electroporation solution to a final volume of 100 microliters and keep the cell DNA mixture on ice. Transfer the cell DNA mixture to the electroporation cuvette, place the cuvette in the electroporator chamber.

Measure the impedance by pushing the appropriate button on the electroporator and ensure that it is 0.030 to 0.055 ohm. Perform electroporation according to the settings indicated in the text protocol. Add 400 microliters of electroporation buffer plus rock inhibitor to the cuvette and then transfer all of its contents to a 1.5 milliliter tube.

Incubate at room temperature for 30 minutes to allow the cells to recover. After 30 minutes, spin at 400 times G for three minutes at room temperature. Remove the supernatant and re-suspend the palette in 20 microliters per well of basement matrix.

Seed approximately one times 10 to the fourth to one times 10 to the fifth cells per well in a 48 well plate and add EGF plus noggin, GSK3 inhibitor, rock inhibitor and 1.25%dimethyl sulfoxide medium. Incubate at 37 degrees celsius. On the following day, change the medium to EGF plus noggin, GSK3 inhibitor and rock inhibitor and check transvection efficiency by observing GFP expression.

Keep organoids at 37 degrees celsius. Two days later, replace the medium with fresh medium. Four days after electroporation, change the medium to WENR plus nicotinamide and rock inhibitor medium and continue to incubate the cells at 37 degrees celsius.

The presence of the correct number of gRNA inserts in the concatemer vector is confirmed by double restriction digestion with EcoR1 and BglII. The expected size of the lower band is approximately 1.6 kilobase for a four gRNA concatemer vector and 1.2 kilobase for a three gRNA concatemer vector. In addition, digestion with Bbs1 confirms that gRNAs have been cloned into the right position and consequently, all Bbs1 recognition sites are lost.

Confirmed constructs are delivered to mouse intestinal organoids by electroporation to achieve transvection efficiency of up to 70%as shown by GFP expression. After watching this video, you should have a good understanding of how to generate gRNA concatemer vectors and how to efficiently deliver them to 3D organoid cultures by using electroporation, in order to achieve knockout of multiple genes at the same time.