This method allows the precise modeling of recurrent leukemogenic gain-of-function mutations in primary human hematopoietic stem and progenitor cells and thereby investigating the role in leukemic transformation. The main advantage of this technique is that the CRISPR-Cas9-based mutation engineering is combined with the introduction of a fluorescent reporter. This allows uniquely the identification, enrichment, and tracking of pure heterozygously-mutated cell populations.
Demonstrating the procedure will be Tommaso Sconocchia, a post-doctoral research fellow from my laboratory. To begin, design the single guide RNA using the online Benchling design tool by selecting the plus create option. Then, click on DNA sequence"followed by import DNA sequences"and import from databases"option.
Then enter the desired gene and select human"as the species. Click on the search"option and select the correct transcript import. Select the region of interest in which to introduce the double strand break.
Then select the CRISPR"option on the right-hand side of the screen, followed by design and analyze guides. Next, select single guide"while keeping the guide length to 20 base pairs and PAM sequence for editing with SP-Cas9. Pick a guide with a high on-target score and a high off-target score and order the single guide RNA as a chemically modified synthetic single guide RNA from a commercial vendor.
To design the HDR template, import the genomic sequence and the coding sequence into software for molecular cloning. From the genomic sequence file, design the left homology arm by selecting, ideally, 400 base pairs on the five prime end of the double strand breaks and paste this sequence into a new file. From the genomic sequence file, design the splice acceptor sequence by selecting the last 150 base pairs of the targeted intron and pasting it after the left homology arm.
From the coding sequence file, select the cDNA of interest. Codon optimize the cDNA and insert it after the splice acceptor sequence. After the cDNA of interest, insert a three prime polyadenylation signal following a promoter sequence for the fluorescent protein.
Then, insert the sequence for the fluorescent protein and a second, but different polyadenylation signal. From the genomic sequence file, design the right homology arm by selecting ideally 400 base pairs on the three prime end of the double strand breaks and insert the sequence after the second polyadenylation. Then, create a copy of the whole template and modify the cDNA of interest so that it contains the sequence of the desired mutation.
Exchange the fluorescent protein with a different fluorescent protein. Construct and clone the HDR templates into the AAV expression vector. For recombinant AAV6 preparation, prepare 10 150 millimeter dishes each containing 11 million HECK293T in 20 milliliters of DMEM supplemented with 10%FBS, 1%penicillin streptomycin, and 25 millimolar HEPES.
Then, place the dishes in the incubator at 37 degrees Celsius, 5%carbon dioxide, for 24 hours. After carefully discarding the old medium, replace it with 20 milliliters of antibiotic-free DMEM supplemented with 10%FBS, 25 millimolar HEPES, and one millimolar sodium butyrate. Next, prepare two 15 milliliter tubes labeled tubes one and two.
To tube one, add five milliliters of reduced serum medium, 60 micrograms of recombinant AAV6-mutated HDR plasmid, and 220 micrograms of PDGM6 helper plasmid. To tube two, add five milliliters of reduced serum medium and 1120 microliters of one milligram per milliliter polyethylene amine solution. After adding the contents of tube two to tube one, vortex for 30 seconds, and then incubate for 15 minutes at room temperature.
Carefully add drop-wise 1.1 milliliters of the solution to each dish and distribute by swirling gently. Place the dishes in the incubator at 37 degrees Celsius, 5%carbon dioxide, for 48 hours. Then add 250 microliters of 0.5 molar EDTA to each dish and place them in the incubator for 10 minutes.
Harvest the cells by washing them off the dish and transfer to a 500 milliliter centrifuge tube. Centrifuge at 2000 G for 10 minutes at four degrees Celsius and discard the supernatant. Loosen the pellet by vortexing and extract the virus by using an AAV purification kit.
After aliquoting the purified virus, store it at minus 80 degrees Celsius. After thawing CD34 positive hematopoietic stem and progenitor cells, transfer the cells into 10 milliliters of pre-warmed RPMI supplemented with 1%penicillin streptomycin. Centrifuge at 350 G at room temperature for 10 minutes.
Suspend the cells in hematopoietic stem and progenitor cell retention medium to a concentration of 250, 000 cells per milliliter and incubate at 37 degrees Celsius and 5%carbon dioxide for 72 hours. Next, harvest the cells in a 15 milliliter tube. Count and check the cell viability by trypan blue exclusion.
To prepare the ribocucleoprotein complex, add 15 micrograms of Cas9 and eight micrograms of single guide RNA in a 1.5 milliliter tube and incubate at 25 degrees Celsius for 10 minutes in a heating block. While the ribonucleoprotein complex is incubating, centrifuge the cells at 350 G for five minutes and discard the supernatant. After suspending the cells in 100 microliters of nucleofection solution, mix the cells with the ribonucleoprotein complex and transfer them into the cuvette.
After gently tapping the cuvette to remove any residual air bubbles, insert the cuvette into the holder of the transfection system and electroporate the cells. Immediately after electroporation, add 400 microliters of pre-warmed hematopoietic stem and progenitors cell retention medium without penicillin streptomycin and transfer the cells with a fine transfer pipette into a culture plate containing pre-warmed hematopoietic stem and progenitor cell retention medium without penicillin streptomycin. Then transfer the plate to the incubator.
Thaw the vials containing the frozen recombinant AAVs on ice and transduce the cells by pipetting the optimal amount of each recombinant AAV6 to the cell suspension. After gently mixing the cell suspension with the pipette, incubate the transduced cells for six to eight hours at 37 degrees Celsius and 5%carbon dioxide. Following six to eight hours, collect the cells in a tube and centrifuge them at 350 G at room temperature for five minutes.
Discard the supernatant and replace it with fresh pre-warmed hematopoietic stem and progenitor cell retention medium supplemented with penicillin streptomycin and transfer the cells to a cell culture plate. Incubate the cells for 48 hours at 37 degrees Celsius and 5%carbon dioxide. To flow sort the engineered cells bearing the heterozygous gain-of-function mutation, harvest the cells in a 15 milliliter tube and centrifuge at 350 G at room temperature for five minutes as demonstrated previously.
Remove the supernatant and suspend the cells in one milliliter of DPBS containing 0.1%PSA and centrifuge again at 350 G at room temperature for five minutes. After discarding the supernatant, suspend the cells in an appropriate volume of DPBS plus 0.1%BSA depending on the cell number as demonstrated previously. Transfer the cells to a sterile fax tube equipped with a cap and add seven AAD or other viability dye to the cell suspension for live/dead cell exclusion.
Sort the live cells that are double positive for the fluorescent reporter proteins into a collection tube containing 200 microliters of hematopoietic stem and progenitor cell retention medium. After centrifuging the sorted cells at 350 G at room temperature for five minutes as demonstrated previously, to a concentration of 250, 000 cells per milliliter for further expansion in culture or use the cells directly for functional assays. Two days following transvection with the ribonucleoprotein complex and transduction with the recombinant AAV6 viruses, the cells were analyzed by flow cytometry.
Four main populations were detected, including cells with no HDR-based genome editing expressing neither the GFP nor the BFP cells positive only for GFP with only the wild type construct integrated, cells positive only for BFP with only the mutated construct integrated, and GFP and BFP double positive cells with both the wild type and mutated sequences integrated. To validate the successful knock-in of the heterozygous calreticulin mutations in/out PCR was performed on genomic DNA extracted from cells incubated with AAV only and from cells incubated with the ribonucleoprotein and AAV with the knocked-in calreticulin wild type and calreticulin deletion sequence. Cells were sorted based on the simultaneous expression of both fluorescence reporters prior to DNA extraction.
After gel electrophoresis, individual bands were excised from the gel and DNA was extracted for subsequent Sanger sequencing. The sequencing results confirmed the successful seamless integration of the wild type and mutated sequences in the heterozygous calreticulin-mutated hematopoietic stem and progenitor cells. The most important thing to remember when attempting this procedure is to carefully select the best-performing single guide RNA, as this will determine the overall HDR efficiency and, therefore, the frequency of successful knock-in of heterozygous mutations.
Following this procedure, the genetically engineered cells can be used for functional in-vitro and in-vivo experiments such as colony-forming unit assays, differentiation assays, and transplantation into immune-deficient mice.
