Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The overall goal of this technique is to genotype CRISPR/Cas9 induced insertion deletion mutant clones in a high-throughput manner. This method can help answer key questions in the genetics field, such as the phenotypic effect of knocking out a particular gene in a SAIL model of choice. The main advantage of this technique is there is a minimal to high throughput screening that is multiflexible.

Though this method can provide insight into human cancer genetics, it can also be applied to the other systems, such as human, stem, and the primary cell, or the cells of non human origin. We first had the idea for this method when we performed short tandem repeats or STR typing assays. To begin the procedure, transfect the chosen cells with a plasmid, co expressing a fluorescent label cas9 and the single guide RNA.

Trypsinize the cells and select about 3, 500 fluorescently labeled clones. Plate the clones in portions of 500, 1, 000, and 2, 000 cells on 10 centimeter dishes in eight milliliters of penicillin streptomycin supplemented growth medium. Incubate the cells in 5%co2 atmosphere at 37 degrees celsius, replacing the medium every five days until single cell colonies are visible to the naked eye.

Then aspirate individual colonies into a 200 microliter pipette with about five microliters of growth medium. Transfer each colony to a separate well containing 200 microliters of growth medium. Repeatedly pipette each mixture up and down to re suspend the cells.

Incubate the cells under the previously described conditions until the cells reach 50 to 90%confluence. Remove excess culture medium from the wells, and add 25 microliters of 0.05%trypsin EDTA without phenol red to each well. Incubate the cells at 37 degrees celsius for 7 minutes.

Then repeatedly pipette the mixtures up and down to re suspend the trypsinized cells. Inspect the wells under a microscope to verify that the cells have detached from the well walls. To prepare replicates of the individual clones, transfer five microliters of each cell suspension to an empty 96 well culture plate.

Add 200 microliters of growth medium to each well. Then load the wells of an empty 96 well PCR plate with 10 microliters of lysis buffer. Transfer another five microliters of each cell suspension to this 96 well plate.

Briefly centrifuge the plate until the liquid settles in the bottom of the wells. Add 200 microliters of growth medium to the remaining 15 microliters of each cell suspension. Store the original suspension and the replicates under the incubation conditions for later verification of the knockout status of the clones.

Then use thermal cycling to completely lyse the cells in lysis buffer. Briefly centrifuge the lysates. Dilute the lysates with 40 microliters of nuclease free water.

Vortex and briefly centrifuge the diluted lysates. Before performing fluorescent PCR, design and test the specificity of several pairs of unlabeled primers on lysed wild-type cells. Identify the optimal primer pair and obtain the fluorophore labeled forward primers and the unlabeled reverse primer.

Be sure to test the specificity of the primers using vortex cells, lyse using the homemade lysis buffer, as results may vary from using purified genomic DNA. Using the labeled primers, perform 20 microliter fluorescent PCR reactions with three microliters of the diluted lysates to amplify the target regions. Assess the size and relative quantities of the PCR amplicons by resolving five microliters of the amplicons on a 1%agarose gel.

To begin sample preparation, use nuclease free water to dilute amplicons of wild-type and CRISPR/Cas9 targeted DNA to an approximate concentration of 2.5 nanograms per microliter. Mix together equal volumes of the diluted wild-type and targeted DNA samples. Load the wells of a 96 well PCR plate with nine microliters of a 29 to one mixture of deionized formamide and dye labeled size standard and one microliter of the mixed amplicons.

Seal the plate tightly and heat the samples at 95 degrees celsius for three minutes with a PCR thermocycler. Immediately after heating, cool the plate on ice for three minutes. Perform capillary gel electrophoresis with a genetic analyzer.

After electrophoresis is complete, import the data into analysis software and plot the data. Verify the quality of the size standard by reviewing the data in the appropriate dye channel. Then export the dye channel values for the un targeted wild-type controls and targeted clones in txt format.

From this file, import the dye identities, sample file names, fragment sizes, and peak height values into spreadsheet software. Calculate the difference in fragment size between the targeted clone and the un targeted wild type control in each sample. Identify the clones with indel mutations that are not multiples of three base pairs.

Serially expand the selected clones from the stored replicates. Perform Sanger sequencing and western blood analysis to determine the clone genotypes and verify the knockout status of the clones. The hepatocellular carcinoma cell line HEPG2 was targeted with a CRISPR/Cas9 construct against the Nap1L1 gene.

The un targeted wild-type alleles and the CRISPR/Cas9 targeted alleles were PCR amplified using primers labeled with green and blue fluorophores, respectively. By using a specifically optimized fluorescent PCR protocol, the amplicons of the targeted clones were obtained in good yield. No interference was observed between the green and blue fluorescent dyes within samples of individual clones.

Analysis of the fragment sizes, determined from capillary gel electrophoresis, revealed the deletion of one and 10 base pairs from the two clones shown here. Sanger sequencing results were consistent with the capillary gel electrophoresis analysis. Western blood analysis confirmed that the selected clones did not express NAP1L1.

While attempting this procedure it's important to remember to label your samples carefully, as there are many samples involved, and also to minimize the exposure time of your fluorophore label amplicon as they are light sensitive. Following this procedure, other methods like quantative RT PCR, western blood analysis, and the Sanger sequencing can be performed in order to answer additional questions like what are the genes of interest, is completely knocked out. After watching this video you should have a good understanding of how to genotype CRISPR/Cas9 induced mutant clones in a high-throughput manner using fluorescent PCR coupled to capillary gel electrophoresis.