This method could help us answer key questions related to gene expression showing us exactly how chromatin structure impacts gene expression and how chromatin organization regulates transcriptional dynamics. We developed this technology because we wanted to be able to controllably alter chromosomal architecture to enable creation of long-range chromatin looping. While at the same time have the ability to reverse the chromatin context to restore the endogenous chromosomal structure.
This method was designed for ease of use and wide applicability. We took advantage of a non-toxic dimerizer as well as orthogonal species of dcas9 in order to allow the system to be used more broadly. We developed this method because we wanted to be able to answer the long-standing chicken-and-the-egg question of whether gene expression begets chromosomal architecture or whether chromosome structure leads to changes in gene expression.
Design of the CRISPR-guide RNA sequences and maintenance of the cell cultures are described in the text protocol. Plasmid maps and the primers utilized are listed there in. Begin the plasmid preparation by mixing five micrograms of the lentiviral CRISPR plasmid with three microliters of BsmB1 three microliters of alkaline phosphatase six microliters of 10x digestion buffer and 0.6 microliters of freshly prepared 100 millimolar of DTT.
Bring the total volume to 60 microliters of double distilled water and incubate the mixture at 37 degrees celsius for 30 minutes to digest and de-phosphorylate the plasmid. Then, gel purify the digested plasmid in the loop plasmid and double distilled water. Next, prepare annealing reactions for the guide RNA pairs.
Combine one microliter of each guide RNA at 100 micromolar with one microliter of 10x T4 ligation buffer 6.5 microliters of double distilled water and 0.5 microliters of T4 PNK for a total reaction volume of 10 microliters. To anneal the guide RNA pairs to their target sequences incubate the mixtures at 37 degrees celsius at 30 minutes followed by an incubation at 95 degrees celsius for 5 minutes then gradually reduce the temperature to 25 degrees celsius by decreasing it by five degrees celsius per minute. Next, dilute the annealed guide RNAs at one to two hundred in double distilled water.
Now, prepare the two ligation reactions. Mix one microliter of the digested plasmid with 0.5 microliters of a diluted perivaneal guide RNAse 2.5 microliters of 2x ligation buffer one microliter of double distilled water and 0.5 microliters of ligase. Incubate the reactions at room temperature for 10 minutes to ligate the guides BsmB1 digested plasmid.
Then transform the newly ligated plasmid into stable three bacteria and amplify the bacteria using any plasmid preparation method. On a six-well plate, per well, seed 750, 000 2n3 Tcells in DMEM with 10%FBS and 1%pen strep. Use one well for each construct and incubate the cells for 24 hours.
The next day, change the medium to fresh antibiotic free-DMEM with 10%FBS. Right after changing the medium for each well of plated cells prepare a tube of Lentivirus production mixture. Per each reaction, dilute 11 microliters of lipid-base transfection reagent with 150 microliters of the Opti-MEM medium for each reaction.
Then in separate tubes prepare the DNA. Combine two micrograms of the CLOud9 lentiviral vector plasmid with two micrograms of the other lentiviral packaging components. Dilute this mixture in 150 microliters of the Opti-MEM medium.
Then combine equal volumes of the diluted lentiviral plasmid mixture in the diluted lipid-based transfection reagent and incubate the mixtures for five minutes at room temperature. Then to each well of cells add one tube of the prepared mixtures and continue the incubation. 48 hours later, transfer the viral production media from the plate wells into conicals and spin them down at 300 g for five minutes.
Then transfer the supernatant to fresh tubes to discard the pelleted debris. Now, immediately use the viral production media to transduce the target cells or freeze it at 80 degrees celsius for future use. Begin with adding 250 microliters of each viral construct of interest, which in this case, is comprised of complementary CLOud9 plasmids to a 50 milliliter conical tube containing 80, 000 cells.
Then add enough Polybrene so that it is between one and eight microgram per milliliter in solution. Then adjust the total volume in each tube to one milliliter using antibiotic-free media. Now to increase the contact between the virus particles and the cells spin the cells at 800 g for 30 minutes at room temperature.
Then re-suspend the cells at the supernatant by pipetting. Next, load the cells'suspension onto culture plates and incubate them for 24 hours. The following day, collect the cells.
Centrifuge them at 300 g for five minutes at room temperature and re-suspend them in normal medium. The next day, add puromycin and hygromycin to the medium to select for doubly transduced cells. The appropriate concentration of antibiotic should be determined in advance for each cell type.
Then, incubate the cells in the selection media for at least three days before proceeding with downstream applications and continue to maintain the cells and selection media. To dimerize the CLOud9 selected and transduced cells add one millimolar ABA to the culture dish. For controls, add DMSO, then maintain the cells with ABA or DMSO for the duration of the experiment.
To reverse the dimerization, wash the cells with enough PBS to cover the surface of the plate twice. Thereafter, culture cells in ABA-free media to maintain them in an undimerized state. Appropriate use of the CLOud9 system induces reversible contact of the complementary CSA and CSP CLOud9 constructs through the addition or removal of ABA to cell culture media.
The CSA and CSP constructs are localized to appropriate genomic regions using standard CRISPR guide RNAse. Chromatin immunoprecipitation followed by quantitative PCR was utilized to ensure the accurate localization in targeting of each CLOud9 component. Additionally coimmunoprecipation with and without ABA verified CSA and CSP dimerization in the presence of the ligand as well as the reversibility in the absence of the ligand.
After ABA dimerization, greater contact between beta-globin and the LCR was measured by chromosome confirmation capture. However, this was not observed in the controls containing either only the CSA or only the CSP construct. Creating the LCR beta-globin interaction did not completely eliminate the endogenous LCR globin contact.
It only added to the original contact. The increased in beta-globin LCR contacts were observed through 72 hours of dimerization regardless of the exact region within the targeted LCR or the beta-globin promoter region. Lastly, the reversibility of the system was confirmed with chromosome confirmation capture after removing ABA.
This resulted in the complete renewal of the endogenous confirmation. While attempting this procedure, it is important to remember to change media and add fresh dimerized CLOud9 transduced cells everyday. Don't forget working with lentivirus can be extremely hazardous and all precautions used at BSL 2 should always be taken when performing this procedure.
