Cis-regulatory elements like promoters and enhancers are key determinants of gene expression. Traditional approaches to study these elements are laborious and often involve the use of heterologous reporter genes. Here we demonstrate the use of CRISPR to examine the transcriptional role of a RUNX1 intronic silencer in the leukemia cell line.
The protocol is simple to perform and allows fast assessments of gene regulatory functions in the endogenous gene context. Hematopoietic cells are often difficult to transfect with a plasmid based methods. In this protocol, we use the electroporation to deliver preassembled Cas9, guide RNA, complexes into the cells.
The advantages of this approach include use of improved editing efficiency and cell viability as well as reduced off-target effects. Also, the subsequent use of fragment analysis enables simple and fast screening of the desired mutant clones from a large amount of samples. Demonstrating the procedure will be Yuk-Lin Yung, technician for my laboratory.
Begin by designing two CRISPR RNAs, one five prime and the other three prime of the target cis-regulatory element, or CRE. Ensure that a PAM of NGG is located immediately downstream of the target sequence for Cas9 recognition. To form the gRNA complex, combine the CRISPR RNA and the tracer RNA in TE buffer according to manuscript directions, for a final duplex concentration of 44 micromolar.
Incubate the mixture at 95 degrees celsius for five minutes and allow it to cool to room temperature. Dilute the recombinant Cas9 nuclease with PBS for a final nuclease concentration of 36 micromolar. Then mix an equal volume of the diluted nuclease with each of the gRNA duplexes and incubate them for 20 minutes at room temperature to allow the RNP complex to form.
Centrifuge 2.5 million cells in a 1.5 milliliter tube at 500 times g for five minutes. Then discard the supernatant and re-suspend the cells in 163 microliters of RPMI 1640 medium without phenol red. Add 16.7 microliters of the RNP complex and 3.6 microliters of 100 micromolar electroporation enhancer to the cells and transfer the mixture to a 0.2 centimeter-gap electroporation cuvette, taking care not to introduce any bubbles.
Electroporate the cells and transfer them to a T-25 tissue culture flask containing 6 milliliters of complete RPMI 1640 medium. Incubate the cells at 37 degrees Celsius with 5%carbon-dioxide. One day after electroporation, dilute the cells to 5000 cells per milliliter in complete RPMI 1640 medium.
And add 100 microliters of the cell suspension into each well of a 96-well tissue culture plate. Culture the cells for seven to 14 days then extract genomic DNA using a high-throughput purification system. Add 100 microliters of plate binding and lysis buffer to each well of a 96-welled extraction plate, followed by 50, 000 cells re-suspended in 10 microliters of PBS.
Mix the well contents by pipetting up and down. Incubate the plate at room temperature for 30 minutes to allow the genomic DNA to bind to the wells. Then carefully aspirate the solution from the wells and wash them with 120 microliters of wash buffer.
Air dry the plate. Add 20 microliters of PCR mix to each well and run PCR according to manuscript directions. Once the amplification is complete, estimate the amount of the product by measuring the concentration of a selected number of samples with a fluorometer.
Dilute all samples to 0.5 nanograms per microliter with nuclease free water. Mix one microliter of the diluted PCR product with 8.5 microliters of deionized formamide and 0.5 microliters of fluorescent dye-labeled size standard in a 96-well plat compatible with the genetic analyzer. Cover the plate with a plate septa and denature the samples at 95 degrees Celsius for three minutes in a thermocycler.
Perform capillary electrophoresis to separate the labeled PCR products and analyze the results on the analysis software. Check the orange icon to view the labeled fragments and the size standard to assess the quality of size calling. Then, check the blue icon to view the labeled PCR products.
Identify the peaks corresponding to wild-type and mutant products and estimate the mutant level in each sample by dividing the area under the mutant peak by the sum of the area under the wild-type and mutant peaks. Select multiple cell pools with high levels of the expected deletions for further serial dilutions. Repeat the DNA extraction, fluorescent PCR, and capillary electrophoresis steps and select clones with mutant levels greater than 95%for subsequent analysis.
Extract total RNA from the selected clones and perform complimentary DNA synthesis. Mix the RNA with a poly-T primer and dNTPs according to manuscript directions and incubate at 65 degrees Celsius for five minutes. Then, place the reaction on ice for at least one minute.
Add ten microliters of cDNA synthesis mix to the reaction. Then incubate the sample at 50 degrees Celsius for 50 minutes and 85 degrees Celsius for five minutes in a thermocycler. After cDNA synthesis, treat the samples with one microliter of RNase H and incubate them at 37 degrees Celsius for 20 minutes.
Design primers and TaqMan probes specifically for individual transcript variance generated from alternative promoters and clone the DNA fragments containing the specific transcript sequences into plasmid DNA. Make a tenfold dilution series of the recombinant plasmids as standard curves for transcript quantification. Prepare 20 microliters of PCR mix for each sample and run the real time PCR according to manuscript directions.
Analyze the results and normalize the copy number of the target transcripts in each sample with a housekeeping gene. This protocol has been successfully used to delete the RUNX1 intronic silencer and the deletion was confirmed with capillary gel electrophoresis. The expected sizes of the wild-type and mutant PCR products are about 500 base pairs and 230 base pairs respectively.
The size of the mutant products can vary among the clones because of indels formed at the cleavage sites. Sanger sequencing was used to confirm the identity of the deletions. The RUNX1 gene contains two promoters, P1 and P2 which produced three major mRNA transcripts:RUNX1c by P1, and RUNX1a and RUNX1b by P2.Real-time quantitative PCR can be used to determine how the deletion of the silencer element affects expression of these transcripts.
A good design of CRISPR RNAs is important for its assess of the experiment. Ensure that the CRSIPR RNA is packaged closely to the intended deletion region. Also, ensure than a PAM sequence is located downstream of the target sequence for Cas9 recognition.
Apart from studying CREs, this strategy can be used for gene location studies and serves as an alternative to RNA interference to examine gene functions. By combining with chromosome conformation capture techniques CRISPR will certainly help decipher the involvements of CREs in the altered genome organization and gene expression linked to various health problems like cancer.
