This method can help answer the question of how HIV provirus integration influences androgynous gene expression, and why certain genomic integration sites are being selected in lately HIV-infected cells. The main advantage of this technique is that in-vitro models for HIV infection can be produced, which carry pro-viral reporters targeted to chosen integration sites using CRISPR/Cas9-based genome engineering. Demonstrating the procedure will be Thomas Walther, a PhD student, and Julia Bialek, a postdoc from my laboratory.
Begin by using UCSC Genome Browser to in-silico-extract the genomic sequence of the desired genomic locus to be targeted. To choose guide RNAs of 20 nucleotides for targeting of the chosen genomic locus, use E-CRISP web tool. Then select, Homo Sapiens Genome Reference Consortium Human Genome:Build 38.
Reference genome as the organism and input 2, 000 base pairs of the genomic sequence, covering the desired genomic locus previously extracted. Start a guide-RNA search using medium application settings with any PAM, any five-prime-base, off-targets tolerate mismatches and excluded introns and CPG islands. From the list with possible guide-RNA designs that appears, select a guide RNA with a high score for specificity and efficiency, which is close as possible to the desired genomic locus to be targeted.
Open the NCBI Blast browser to blast the chosen guide-RNA sequence against the reference genome. To check for uniqueness of the selected guide-RNA binding site, first select human as the genome and then input the guide-RNA sequence as the query sequence. Then select highly similar sequences or Mega Blast as the program to make sure that the guide-RNA sequence is unique.
After that, select in-silico 1, 000 base pairs, upstream and downstream of guide-RNA sequence from genomic sequence extracted at the beginning. Use these selected 1, 000 base pairs, upstream and downstream as five-prime and three-prime homology arms for targeting vector construction and adjoin reporter sequence to be targeted. 24 hours prior to transfection of jurkat T-cells, prepare one complete 6-well plate of the cells for a single targeting experiment.
To start, plate 1, 250, 000 cells in 2.5 milliliters of RPMI 1640 with supplements and without antibiotics, per well. On the following day, add to micrograms of circular targeting vector and two micrograms of Cas9 and guide-RNA expression plasmid per well, to 250 microliters of commercial RPMI medium optimized for transfection, in a reaction tube and mix well by vortexing. Then slowly add 12 microliters of transfection agent to the DNA medium without touching the wall of the tube.
Flick the tube and incubate for 15 minutes at room temperature. Add the mixture dropwise to one well of cells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide.
And 72 hours after transfection, pull the transfected cells, count them and prepare for enrichment by FACS. After confirming the targeting events in the mixed targeted cell population, start generating single cell clones by preparing jurkat-conditioned medium in advance, by collecting RPMI with antibiotics from healthy, untreated jurkat T-cells. Centrifuge the tubes for 5 minutes at 300 G, and filter the supernatant with a 25 milliliter syringe, using a 0.22 micrometer filter into new, 50 milliliter conical tubes.
Count the targeted cells previously enriched by FACS, 10 to 14 days after expansion, and then dilute them in RPMI with antibiotics to a concentration of 100, 000 cells per milliliter. Dilute 100 microliters of this cell dilution with 9.9 milliliters of medium to achieve 1, 000 cells per milliliter. Then dilute one milliliter of this dilution with nine milliliters of medium to a concentration of 100 cells per milliliter.
To plate 96 well plates to contain one cell per well, gently mix one milliliter of 100-cells-per-milliliter solution with five milliliters of condition medium and four milliliters of fresh medium, in a sterile reagent reservoir. Then, use a multi-channel pipette to add 100 microliters of the respective cell dilution per well of new 96-well round bottom plates to achieve one and two cells per well dilutions. Stack the 96 well plates and cover each stack with a six-well plate containing three milliliters of PBS per well.
Incubate the plates at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide, for three weeks. After completed incubation, visually confirm the presence of grown colonies using light microscopy with four-times magnification, and mark the wells with grown colonies. Gently resuspend cells of one marked well by pipetting.
Transfer 100 microliters of this cell suspension into one well of freshly prepared 96-well round bottom plate with 100 microliters of RPMI with antibiotics, per well and repeat for all marked wells, mix gently by pipetting. To duplicate the plate, transfer 100 microliters of this cell suspension into a second empty, 96-well round-bottom plate and repeat this process for all marked wells. Finally, fill the blank wells with 200 microliters of RPMI with antibiotics.
Incubate the plates at 37 degrees Celsius and 5%carbon dioxide. After the duplicate plate has incubated for 24 to 48 hours duplicate it again by adding 100 microliters of RPMI with antibiotics to every well. Use a multi-channel pipette to mix gently and transfer 100 microliters to each well of a new 96-well round-bottom plate.
Place one of these two duplicate plates in the incubator. Use the second of the new duplicate plates for flow cytometry, by first adding five microliters of PMA/Ionomycin Master Mix per well, to stimulate the cells. Incubate for 24 hours and prepare the cells for flow cytometry as described in the text.
Gate any viable single cells based on size in forward and sideward scatter. Analyze fluorescent reporter gene expression by flow cytometry. To screen single-cell clones by PCR, design primers P5 and P6 for screening PCR based on the chosen reporter sequence, to amplify 500 to 800 base pairs of the reporter sequence.
For positive control PCR, design primers P7 and P8 that amplifies 630 base pairs of a wild-type, non-targeted genomic locus. Design a third primer pair that amplifies 500 to 600 base pairs of the targeting-vector backbone as a control for unspecific integration of targeting-vector backbone sequences. Once the clones in the second duplicate plate have grown sufficiently, prepare them for PCR screening by first centrifuging the plate for 10 minutes at 300-G at room temperature.
Carefully take off the supernatant, making sure not to disturb the cell pellet. Wash the cells with 100 microliters of PBS by gentle pipetting, centrifuging the plate for five minutes at 300 G at room temperature. Remove the PBS and add 200 microliters of lysis buffer per well.
Pipette gently to mix, and transfer the suspension to a new PCR plate. Seal the plate with paraffin film and incubate for one hour at 55 degrees Celsius in a thermocycler. After centrifuging down cell debris at maximum speed for 10 minutes, transfer the supernatant to a new PCR plate.
Add 10 microliters of this cell lysate to each well of a 96-well PCR plate filled with 110 microliters of distilled H2O, per well. Then incubate for 10 minutes at 99 degrees Celsius in a thermocycler, to inactivate proteinase K.Use 2 microliters of this cell lysate as a template and use a commercial PCR master mix for screening, control and backbone PCR. Run PCR for 38 to 40 cycles of PCR amplification in a 96-well format.
Use five microliters of PCR products and run them on a 1.5%agarose TAE gel to analyze and combine the flow cytometry and PCR results, to confirm single cell clones. Reporter gene expression after PMA/Ionomycin induction was confirmed by both flow cytometry and PCR in four to 12%of cells, depending on integration site. PCR analysis of genomic DNA using primers for five-prime and three-prime integration junction, confirm that targeting events have occurred.
After the screening of single cell clones for correct targeting by flow cytometry, clones with high, low and no fluorescent reporter gene expression were observed. Single cell clones were also confirmed by PCR using reporter-specific primers and control primers. Clones confirmed positive in screening PCR were expended and further analyzed for correct targeting by Southern blot, using a reporter-specific probe and a genomic-probe binding outside the reporter.
Only a portion showed correct band sizes in Southern blot analysis and had heterozygously integrated the reporter, showing that it is important to verify PCR results by Southern blot. Clones were also analyzed by sequencing to further confirm positive single-cell clones. Sequencing of the target site:homologous allele, were the reporter had not integrated, revealed Cas9-induced mutations.
Once mastered, generation of HIV reporter-lines can be accomplished in two to three months. Prior to targeting, it's important to spend enough time choosing an integration site at and appropriate reporter, depending on the research question you would like to tackle. Reporters can for example, include different HIV regulatory elements if desired.
Following this procedure, cell lines can for example, be used to test the effect of different latency rising agents on the transcriptional activity of the reporter LTR, depending on its integration site. After watching this video you should have a good understanding of how to generate integration-site-specific HIV reporter-lines for designing guide RNAs and targeting vector, performing CRISPR/Cas9-based genome engineering in jurkat cells, producing single cell clones and selecting for correctly targeted clones by FACS and PCR screening. Don't forget, that if you choose to work with a replication-competent HIV reporter, you need to work under BSL3 safety conditions.
