Generation of Centromere-Associated Protein-E CENP-E^-/- Knockout Cell Lines using the CRISPR/Cas9 System

Genetic deletion of Kinesin-7 and CENP usually results in cell cycle arrest and cell death. The construction of stable CENP knockout cell lines is an important unresolved issue in CENP biology. In this study, we generated CENP knockout HeLa cells using a CRISPR/Cas9 system, and established three optimized screening strategies.

Recently, several genome editing technologies, including zinc-finger nucleases, transcription activator-like effector nucleases, and how we make our nucleases, has been engineered to cleave genomes at a specific size. The CRISPR/Cas9 gene knockout technology has revolutionized basic biology, biotechnology and medicine. CENP is essential for attaching kinetochores to microtubules and aligning chromosomes.

Disrupting CENP, whether through antibody microinjection, sgRNA deletion, chemical inhibition, or genetic deletion, result in chromosome misalignment, mitotic defects, and often cell death, complicating the study of CENP protein mechanisms. In this study, three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment of phenotypes, and the fluorescent intensities of CENP proteins, which improved the screening efficiency and the experimental success rate. Importantly, CENP deletion results in chromosome misalignment, the abnormal location of BubR1 proteins, and mitotic defects.

The interaction modes and the molecular mechanisms of CENP and its inhibitors are important research fields in cell biology and cancer research. In this study, the CENP knockout HeLa cell line has been established as a valuable tool for the validation of the specificity and the toxicity of CENP inhibitors. To begin, add 0.25%trypsin EDTA solution to the HeLa cells and incubate at 37 degrees Celsius for two to three minutes.

Gently dissociate the cells by pipetting and seed them in new plates at a ratio of 1:4 for cell passage. To transfect the plasmid into HeLa cells, mix one microgram of validated PX 4 58 sgRNA plasmid with 50 microliters of reduced serum medium in tube A.In tube B, gently mix two microliters of the transfection reagent and 50 microliters of reduced serum medium and incubate at room temperature for five minutes. Then, combine the contents from tubes A and B and incubate at room temperature for another five minutes.

Add the mixture to the 12-well plate and incubate the cells at 37 degrees Celsius for six hours. At the end of the incubation, replace the medium with fresh DMEM medium. After 24 or 48 hours, assess the transfection efficiency by examining the HeLa cells under a fluorescence microscope.

Dissociate the transfected HeLa cells fully using 0.25%trypsin EDTA and incubate at 37 degrees Celsius for three to five minutes. Next, count the cells using a Neubauer chamber or an automated cell counter. Plate the cells into three separate 96-well plates for each transfected population following serial dilution methods.

Return the cells to a humidified incubator for one to two weeks. For phenotype-based screening and validation of CENP-E knockout, dissociate and expand the cells in 24-well or 12-well plates for five to seven days. Screen the mutant cells with smaller colony diameters using an inverted microscope with 10 times and 20 times magnification objective lenses.

Next, harvest the cells for DNA extraction by employing the column animal genomic DNA extraction kit following the manufacturer's instructions and set up the polymerase chain reactions. Ligate the target DNA into the pMD18-T vector as directed by the manufacturer's protocols. Transfect the ligated plasmid into competent DH5-alpha cells and proceed with the culture for clone selection.

To identify the types of CENP-E knockout, isolate the plasmid DNA with the help of a plasmid extraction kit following the manufacturer's guidelines. Next, carry out Sanger sequencing for the plasmid DNA of each colony using M13 forward and reverse primers. Seed the wild type and CENP-E mutant HeLa cells on 12 millimeter glass cover slips in a 24 well plate.

Remove the complete DMEM medium and fix the cells using a 4%paraformaldehyde and PBS solution at room temperature for 10 minutes. Next, stain the nuclei with DAPI at room temperature for five minutes. Observe and validate the CENP-E mutant cells based on chromosome alignment phenotype using a fluorescence microscope.

The phenotypic screening of the colonies showed that the CENP-E knockout cells showed increased chromosome misalignment compared to the control group. To begin, take a plate of wild type and CENP-E mutant HeLa cells growing on a cover slip. Fix the cells with 4%paraformaldehyde and PBS fixative solution at room temperature for 10 minutes.

Then immerse in PBS for two rounds of five minutes each. Next, permeabilize the cells with 0.25%Triton X-100 and PBS at room temperature for 10 minutes. Immerse them in PBS for two rounds of five minutes each.

Proceed to block the cells with 1%BSA PBS with 0.1%Tween 20 at room temperature for one hour. Dilute the primary antibodies in 1%BSA and PBST and incubate the samples at four degrees Celsius for 12 hours. Discard the primary antibody solutions, then rinse the cells three times for five minutes each with PBS.

Add diluted secondary antibodies to the cell and incubate at room temperature for two hours. Next, discard the secondary antibodies, then rinse the cells with PBS three times for five minutes each. Stain the nuclei using DAPI at room temperature for five minutes.

Mount the slides with the mounting medium and seal them with nail polish. Observe the fluorescence images using a scanning confocal microscope fitted with a 63x magnification 1.40 numerical aperture objective and record them for further analysis. Immunofluorescence staining of the control and CENP-E mutant groups showed that the fluorescent intensities of the CENP-E proteins were completely knocked out in the CENP-E mutant clone 1 cells and significantly decreased in CENP-E mutant clone 3 cells.

The ratios of metaphase cells with chromosome misalignment increase in clones 1 and 3 compared to the control cells. Meanwhile, the ratios of metaphase cells increased significantly in clone 1 cells and clone 3 cells. In addition, the ratio of interphase cells slightly increased in clone 1 and 3 groups after CENP-E deletion compared with the control group.

The rates of prophase, anaphase, and telophase cells were slightly decreased after CENP-E deletion, whereas the rates of metaphase cells increased after CENP-E deletion. To begin, take the wild type and CENP-E mutant HeLa cell cultures and add 300 nanomolar colchicine before incubating them for five hours. Next, incubate the cells with 0.25%trypsin EDTA at 37 degrees Celsius for three minutes, and collect the cells into 1.5 milliliter centrifuge tubes.

Next, centrifuge the cells at 1000 G for five minutes at room temperature. Discard the supernatants and add 1.2 milliliters of 0.075 molar potassium chloride solution. Incubate the cells at 37 degrees Celsius for 20 minutes.

At the end of the incubation, centrifuge the cells, discard the supernatant, and add 0.2 milliliters of fixative solution for prefixation. Mix gently for one minute, then collect the cell pellets as demonstrated earlier, and add 1.5 milliliters of fixative solution at room temperature for 10 minutes. After centrifuging the cells and discarding the supernatant, add 0.6 milliliters of the fixative solutions to create a cell suspension.

Carefully drop three to five droplets of the cell suspensions from 35 to 40 centimeters onto the ice slides. Immediately dry the slides using an alcohol lamp and stain them with 10%Giemsa sustaining solution for seven minutes. Rinse the slides with running water for two minutes, then examine the samples using a light microscope equipped with a Plan Fluor 40x magnification, 0.75 numerical aperture objective.

The karyotype analysis revealed decreased chromosome numbers in the CENP-E mutant HeLa cells compared with the wild type cells.