This protocol uses a high throughput CRISPR gene editing workflow to molecularly dissect micro RNA genes organized and clustered units. And to determine how these noncoding RNA networks coordinate cancer progression pathways. This CRISPR gene editing procedure allows the investigator to quickly generate an entire panel of cell lines carrying unique micro RNA cluster deletion combinations while avoiding time consuming DNA vector sub cloning.
This method will provide a clearer understanding of how clustered micro RNAs function cooperatively to regulate tumor growth, aggressiveness, and drug resistance before translating micro RNAs into the clinic as therapeutic and diagnostic tools. Demonstrating the procedure will be Grace Yi, a research assistant in my laboratory. To begin, create a DNA file containing the complete genomic sequence of the micro RNA cluster region of interest in at least one kilobytes of surrounding genomic regions using a DNA sequence annotation software program.
Next, design four unique CRISPR RNAs for each targeted micro RNA locus. Two designed CRISPR RNAs to target complimentary DNA sequences immediately five prime of the micro RNA hairpin cluster and two designed CRISPR RNAs to target complimentary DNA sequences immediately three prime of the micro RNA hairpin cluster. Use a feature editing tool in the created DNA file to mark the DNA target sequence for each designed CRISPR RNA to be synthesized.
Designed and synthesized PCR primers flanking the targeted micro RNA cluster regions for genotyping CRISPR cell lines. Perform a doxycycline concentration curve on newly generated lenti inducible Cas9 cell lines to determine the optimal conditions for Cas9 protein induction. Plate five times 10 of the fourth cells per well of each six well plate and grow at 37 degree Celsius, 5%carbon dioxide, in the preferred medium for 24 hours.
Dilute dox in the preferred medium with a final dox concentration curve ranging from 0 5100, 150, 250 to 500 nanograms per milli liter. Label the wells of each seated six well plate from one to six. Remove the medium and replace with the appropriate dox concentrations.
Grow plates one to four until 24 hours, 48 hours, and 72 hours dox induction and 120 hours post dox withdrawal. Harvest the wells of the plate at each time point. Process the cell pellets and ripa buffer for lysate isolation.
Perform western blot analysis with 40 micrograms of protein to measure Cas9 protein induction and determine optimal Cas9 concentration. On paper, map out the five prime and three prime CRISPR RNA pair combinations that will be transected into each well of a 24 well plate targeting the entire micro RNA cluster, various clustered micro RNA gene combinations, as well as individual micro RNA cluster members. Plate the lenti inducible cast nine cell line at five times 10 to the fourth cells per well of a 24 well plate in the preferred medium containing the optimized dox concentration.
Grow the cells for 24 to 48 hours at 37 degrees Celsius, 5%carbon dioxide to induce Cas9 protein expression. Transfect the lenti inducible Cas9 cells with a prepared five prime and three prime guide RNAs. Label four 1.5 milliliter micro centrifuge tubes per targeted micro RNA locus.
In each tube, mix one to one molar ratio of tracer RNA and unique CRISPR RNAs together to form the two micromolar guide RNA complex. Add 2.5 microliters of tracer RNA, 1.25 microliters of the five prime positioned CRISPR RNA, 1.25 microliters of the three prime positioned CRISPR RNA, and five microliters of 10 millimolar tris hcl pH 7.5 buffer to a 1.5 milliliter centrifuge tube. Micro centrifuge for 30 seconds at 16, 000 times G to mix.
Incubate at room temperature for five minutes. To each tube at 40 microliters of reduced serum medium to the 10 microliter of guide RNA complex reaction. Mix gently by pipetting up and down.
In a clean 1.5 milliliter centrifuge tube, mixed two microliters of the transfection reagent and 48 microliters of reduced serum medium gently by pipetting up and down. Incubate at room temperature for five minutes. To each tube containing the 50 microliters of guide RNA mix, add the 50 microliters of the diluted transfection reagent.
Pipette the mixture slowly up and down once to mix. Incubate at room temperature for 20 minutes. Add 400 microliters of antibiotic free medium supplemented with doxycycline to each tube containing the 100 microliters of the guide RNA transfection mix.
Pipette gently to mix. Remove the medium from the doxycycline induced lenti inducible Cas9 cells. Add 500 microliters of media doxycycline guide RNA transformation mix based on the 24 well plate experimental map.
Incubate the transfected cells for 48 hours at 37 degrees Celsius in 5%carbon dioxide. Replace the medium with the fresh prepared medium without doxycycline. Allow the cells to recover for another 24 to 48 hours at 37 degrees Celsius and 5%carbon dioxide.
Harvest the transfected cells and prepare for genotyping and single cell delusions. Separate the 150 microliters of resuspended cells into three parts. Freeze one third of the transfected cells in medium plus 10%DMSO in cryo vials for long term storage.
Transfer one third of transfected cells into a clean 0.2 milliliter PCR tube for genotyping. Prepare the final one third of transfected cells for counting and dilution in a 96 well plate format to generate single cell colonies. Using a 12 channel multi pipetter and a sterile reagent reservoir, add 100 microliters of diluted cells per well to two rows of the plate for each dilution.
Allow four to six weeks for the cells to grow to cofluency for single cell colony expansion. Collect approximately 10 to 15 single cell colonies for genotyping. Identify at least three independent knockout single cell colony lines for each targeted micro RNA locus.
Retain wild type single cell lines as controls. Resuspend been the cell pellet in four microliters of five X DNA polymerase buffer, one microliter of proteinase K, one microliter of RNase A, and nuclease free water up to a total volume of 20 microliters. Lice the cells in the thermo cycler at 56 degrees Celsius for 30 minutes and 96 degrees Celsius for five minutes.
Store the cell lysates at minus 20 degrees Celsius until ready for PCR genotyping. Perform a PCR genotyping reaction using the designed PCR primers that flank the guide RNA five prime and three prime guide RNA targeted sites. Run the PCR reaction in a thermocycler using the program mentioned in the text manuscript.
Load the PCR products onto a 1%agarose gel for electrophoresis analysis. Extract DNA from the isolated PCR fragments of the predicted molecular size for the knockout genotypes. Prepare the samples for DNA sequencing.
Validate that the CRISPR reaction was successful and perform DNA sequencing of the isolated PCR fragments to identify the Cas9 cleavage site and confirm micro RNA locus deletion. Unique CRISPR RNAs were designed that targeted the entire 35 kilo bases miR-888 cluster region. Smaller cluster combinations within the miR-743 family or the miR-891 family as well as micro RNA deletions.
Gel electrophoresis analysis of the PCR reactions indicated that the predicted DNA fragment size representing the knockout genotype was amplified for each CRISPR transfection. Single cell colonies were genotyped by PCR in sequence verified. DNA sequencing of these isolated knockout PCR fragments confirmed that the transfected five prime and three prime guide RNAs directed Cas9 cleavage ligation approximately three nucleotide upstream of the PAM sites and validated genomic loss of the targeted micro RNA locus.
WST-1 proliferation assays comparing miR-891a knockout and wild type cells confirm that overexpression of micro RNA mimic lentiviral vector induces prostate cell growth and therefore it was predicted that miR-891 a loss would show reciprocal effects. Besides careful guide RNA design it is important to quickly generate single cell colonies through each micro RNA knockout line. It is especially relevant if the micro RNA knockout cells have reduced growth of viability and would be easily outcompeted in mixed populations with wild type cells.
Additional methods such as quantitative realtime PCR, northern blot, and RNA sequencing should be performed to confirm that the CRISPR knockout cell lines do not express mature micro RNA for the deleted locus.
