This video will demonstrate a simple method for investigating multiple candidate genes in AML in parallel, using the Crisper-Cas9 system combined with the flow cytometry based competitive growth assay. The main advantage of this technique is that it is rapid and scalable. Demonstrating the procedure will be Bo Rui Chen, a post doc from our lab.
A web based CRISPR design software is used to design four to six single guide RNAs for the gene of interest. To begin, fill in the appropriate information in the fields with asterisks. Then select the target genome for single guide RNA design.
Paste the target sequence in the sequence box, and click on the submit button. For cloning in the desired single guide RNA expression plasmid, prepend the nucleotides CACC to the sense oglionucleotide and AAAC to the reverse complimentary antisense oligonucleotide. Premixed sense and antisense oligonucleotides are then ordered from a company in a 96 well plate, labeled Oligo Mix.
Set out a separate U-Bottom 96 well plate, labeled Annealing Plate. Prepare a master mix of one microliter of T4 polynucleotide kinase, one microliter of 10x T4 DNA ligation buffer, and six microliters of water per annealing reaction. Add 8 microliters of the master mix to each well of the Annealing Plate, followed by 2 microliters of premixed sense and antisense oligonucleotides.
Pipette two to three times gently to mix well, and then spin the plate briefly to get the mixes to the bottom of the wells. Use the following annealing program in a PCR machine. 37 degrees Celsius for 30 minutes, 95 degrees Celsius for two minutes and 30 seconds, followed by slow cooling to 22 degrees Celsius at a rate of 0.1 degrees Celsius per second.
When the annealing is complete, prepare a fresh 96 well plate, labeled Diluted Oligo Mix, by diluting the phosphorylated and annealed oligonucleotides from the annealing reaction one to 200 with water. Linearize the single guide RNA expression vector by digesting five micrograms of the vector with 1.5 microliters of BBS1 restriction enzyme, using an appropriate buffer at 37 degrees Celsius for two hours. Next, take a fresh 96 well plate labeled Ligation Plate, and add 20 nanograms of the BBS1 digested single guide RNA expression vector to one well per desired single guide RNA ligation.
To this, add 2 microliters of phosphorylated annealed oligonucleotides from the diluted Oligo Mix plate. Add to each well, one microliter of 10X T4 ligase buffer, and one microliter of T4 ligase enzyme, and gently pipette to mix. Incubate the plate at room temperature for two hours.
About 10 minutes before the ligation reaction is done, Fill 90 microliters of chemically competent E.coli cells on ice. Transfer 10 microliter aliquots of the competent cells into each well of a separate 96 well plate individually. Add the ligation mixture into the wells containing the competent cells.
Pipette up and down gently, and incubate at room temperature for 10 minutes. Pipette five microliters of the bacteria DNA mix from each transformation reaction directly into a well of a 6 well plate, containing LB agar with 100 milligrams per milliliter of ampicillin. Add approximately five to eight glass beads to each well, and shake the 6 well plate eight to 10 times in a circular motion.
Incubate the plates at 37 degrees Celsius overnight. On the following day, pick one to two single colonies from each well, with a sterile 20 microliter pipette tip, and streak it onto a labeled spot on a sterile 10 centimeter petri dish, with LB agar and 100 milligrams per milliliter of ampicillin After streaking onto the LB plate, eject the tip into three milliliters of ampicillin medium in a 14 milliliter round bottom tube, marked with the corresponding bacterial clone number. The bacterial plate is then sent directly for Sanger sequencing.
After a sequence confirmation of the cloned single guide RNAs, purify the DNA from the corresponding 14 milliliter tube, using a miniprep kit according to the manufacturer's instructions. Single guide RNA lentiviral particles are produced in a 96 well format, as described in the text protocol. And the viral supernatant is immediately frozen at minus 80 degrees Celsius.
One day prior to the transduction of single guide RNAs in AML Cas9 cells, coat a flat bottom non tissue culture treated 96 well plate, with 100 milliliters of recombinant human fibronectin fragment at a concentration of 10 micrograms per milliliter. Wrap the plate with cling wrap to avoid evaporation loss, and leave it on the bench overnight. On the following day, remove the recombinant human fibronectin fragment from each well of the 96 well plate.
Thaw the viral supernatant of each single guide RNA at room temperature. Add 50 microliters of viral supernatant from each tube to each coated well of the transduction plate. Wrap the plate with cling wrap to avoid potential contamination during centrifugation.
Spin the plate at 1, 300 times g at 35 degrees Celsius for 90 minutes. Towards the end of the spinfection, count the high Cas9 expressing clone B3 cells from the culture flask. After counting, spin down the required number of cells, and resuspend in fresh medium.
After the 90 minute spinfection, use a multichannel pipette adjusted to 50 microliters to remove the supernatant from all the wells slowly by tilting the plate. Add 100 microliter of the culture of clone B3 cells to each well slowly, sliding down from the rim. Spin the plate at 1, 300 times g at 35 degrees Celsius for two minutes to let the cells settle to the bottom.
Transfer the plate to a 37 degrees Celsius tissue culture grade incubator. On the following day, add 100 microliters of fresh medium to each well containing single guide RNA transduced cells, such that the final medium volume is 200 microliters. Return the plate to the incubator.
72 hours post transduction, check the percentage of single guide RNA containing BFP positive cells in each well by flow cytometry. Use a cell viability dye to mark and exclude dead cells from the analysis. Continue re-plating a proportion of the cells into new wells with fresh medium after every facts analysis to avoid overgrowth during the assay.
Every two to three days, repeat the facts analysis to check the relative proportion of BFP positive cells compared to the BFP negative counterparts. Analyze the percentage of BFP positive cells for each time point, using a facts analysis software. The key to the success of the competitive proliferation assay is to use the proper gating techniques to ensure that the eventual live cell numbers are accurate.
Drag FCS files for each sample into the software. Double click on any one sample file, and plot a dot plot of forward scatter versus side scatter, with forward scatter on the x-axis, and side scatter on the y-axis. Gate all the cells.
Double click on the gated cells, and plot viability stain on the y-axis versus forward scatter height or area dot plot. Gate the viability stain negative cells. Double click on the gated live cells, and plot BFP on the y-axis versus forward scatter on the x-axis.
Gate the BFP positive cells. Apply all these gates to all the samples by dragging the selected sample to all samples under the group tab. Click on the table editor to make an analysis table of all the samples.
Drag the BFP positive count from one sample to the table, and click on the display button in the table editor to create a batch report of all the samples, with the table displaying the percentage of BFP positive cells. Save the table as an excel file. This system was used to investigate the role of genes that may play a role in the proliferation or survival of human and murine AML cell lines.
Targeting the AAVS1 safe-harbor locus with two separate single guide RNAs had no effect on the proliferation of human MOM13 Cas9 cells In contrast, when the DNA replication associated gene, RPA3, was targeted, a progressive and significant decline in the percentage of BFP positive cells compared to the BFP negative untransduced counterparts was observed. Similarly, the effects of single guide RNAs targeting Dot1l, an epigenetic regulator, and Rhodopsin, the eye pigmentation gene, were tested in mouse MLL-AF9 Cas9 cells. Single guide RNAs targeting Dot1l, showed a dramatic and progressive decline in competitive proliferation in contrast to single guide RNAs targeting Rhodopsin.
These results demonstrate the vulnerability of the MLL-AF9 expressing mass leukemia cells to Dot1l depletion, confirming previously published results. Assuming four to six guide RNAs per gene, this method is well suited to be used for testing at least 16 to 24 genes in parallel, and it can also be applied to test gene dependencies in any cancer cell line. In the case of a larger number of genes, such as an entire molecular pathway that needs to be tested in AML cells, pool CRISPR-Cas9 based screens will be more useful.
