The overall goal of this protocol is to achieve fast and efficient gene disruption through CRISPR/Cas9 and primary hematopoietic progenitor cells using a ribonucleoprotein approach. This method can answer key questions in the fields of normal and malignant hematopoiesis, such as what are the molecular and phenotypic consequences of the loss of gene X.The main advantage of this technique is that it is extremely easy, fast, and cost-effective. The estimated time needed from the experiment conception to the execution is shorter than one week.
Though this method has been optimized for hematopoietic progenitor cells, it can be applied to other cell types, such as primary T-cells, mouse and human hematopoietic cell lines and primary leukemia samples. We first had the idea for this method when we realized how other CRISPR/Cas9 delivery approaches, including plasmids and viruses, were either toxic to our cells or had slow turnaround times. In order to design single-guide RNAs of interest, first navigate to the CRISPRscan website.
Based on the cell of interest, hit the Mouse or Human button and then enter the gene of interest in the UCSC search box and click go. Next, zoom in on the exon that you wish to target. Check if any single-guide RNA target sequences are listed under the CRISPRscan predictions on Genes heading.
Next, click on a target single-guide RNA labeled as a green rectangle and copy the full sequence displayed under the Oligo sequence. Then paste the sequence in a text document. Next, add the nucleotides ATAGC to the three prime end of the sequence.
Place the order for the full length sequence. Now, to synthesize the DNA template for the single-guide RNA, dissolve and then dilute the single-guide RNA forward and universal reverse primers. Then add all the reagents in a PCR tube to perform the overlapping PCR.
After constituting the PCR reaction, place the tube in a thermocycler and run the program. Once the program is over, purify the PCR product using DNA purification columns. Dilute the DNA in 11.5 microliters of elution buffer.
Then, use elution buffer as blank on the spectrophotometer. Measure the concentration of the sample DNA on the spectrophotometer. First, mix the eluted DNA, deoxynucleotide phosphates, reaction buffer and T7 RNA polymerase enzyme mix in the PCR strip tubes.
Then incubate the sample at 37 degrees Celsius for four hours. Next, apply the RNase cleaning agent to remove RNase from the gloves in use. Then, adjust the volume of each RNA sample to 50 microliters with nuclease-free water.
Purify the RNA following the manufacturer's instructions. Finally, elute the RNA in 50 microliters of nuclease-free water. First, use the nuclease-free water to blank the spectrophotometer and then measure the concentration of the eluted RNA.
Use trypan blue exclusion to count the number of cells. For each replicate, collect 200, 000 viable cells per replicate, then add phosphate buffered saline to wash the cells. Then spin the tube at 300 times g for seven minutes.
Post-centrifugation, carefully remove the supernatant. During the course of centrifugation, incubate 1.5 micrograms of Cas9 with one microgram of total single-guide RNA in PCR tube for 20 to 30 minutes. This is to prepare the Cas9-sgRNA RNP complexes for each replicate.
After calculating the volume of the resuspension buffer T required, dissolve the cell pellet obtained in the resuspension buffer T.Then add 10 microliters of the dissolved cell suspension in the PCR tube with the Cas9-sgRNA RNP complexes. Next, switch on the electroporation unit. Slide the cuvette in the cuvette holder.
Then add three milliliters of buffer E.Set the conditions on the electroporation device. To perform the electroporation, first hold the electroporation pipette then extend the claw and grab the disk inside the pipette tip, then firmly press the pipette down to secure the tip. Next, mix the sample by pipetting it up and down 10 times.
Draw 10 microliters of the sample and ensure that there are no air bubbles. Next, insert the tip directly into the electrolytic buffer in the electroporation cuvette. Then hit Start on the screen.
Let the complete message appear on the screen. Then remove the pipette from the cuvette. Next, dispense contents from the cuvette into the well filled with new HSPC culture medium.
To study the efficacy of the Cas9-sgRNA RNP electroporation, HSPCs isolated from the mice are electroporated with single-guide RNase targeting either GFP or tdTomato. These HSPCs ubiquitously expressed GFP or tdTomato. Flow cytometric analysis shows approximately 75 and 74%loss of GFP or tdTomato expression in the HSPCs at the protein level.
Then, T7 endonuclease one based assay is performed with PCR amplified DNA from the GFP locus of the electroporated cells. This is to quantify the gene disruption efficiency of GFP. ImageJ software is used to calculate the ratio of the intensities of the cleaved to the uncleaved bands to calculate the percentage of cleavage.
The efficiency of three different single-guide RNAs targeting human CD45 is tested in the HL-60 AML cell line. Flow cytometry shows single-guide RNA1 as the most efficient one with 98%knockout efficiency when compared to the control. Then, CD45 knockout is also performed in primary human cord blood CD34 positive HSPCs using single-guide RNA1.
Flow cytometry analysis shows almost 80%cells with loss of CD45 with no alteration in CD34 expression in comparison to only Cas9. In order to study engraftment of human CD45 knockout HSPCs, bone marrow and spleen cells are harvested from CD34 positive retro-orbitally transfected NSG mice for analysis, showing edited cells as HLA-ABC positive and CD45 negative. Once mastered, this protocol can be completed in three days, if performed corrected.
While attempting this procedure, it is important to remember to keep all surfaces and reagents RNase-free, verify efficient gene disruption with multiple techniques, and use the proper experimental controls. After watching this video, you should have a good understanding of how to design, synthesize, and transfect Cas9-sgRNA ribonucleoproteins into hematopoietic progenitor cells to obtain fast and efficient gene disruption.
