We thank all members of the Cytoskeleton Laboratory at Fujian Medical University for helpful discussions. We thank Jun-Jin Lin, Zhi-Hong Huang, Ling Lin, Li-Li Pang, Lin-Ying Zhou, Xi Lin, and Min-Xia Wu at Public Technology Service Center, Fujian Medical University for their technical assistance. We thank Si-Yi Zheng, Ying Lin, and Qi Ke at the Experimental Teaching Center of Basic Medical Sciences at Fujian Medical University for their support. This study was supported by the following grants: National Natural Science Foundation of China (grant number 82001608 and 82101678), Natural Science Foundation of Fujian Province, China (grant number 2019J05071), the Joint Funds for the Innovation of Science and Technology, the Fujian Province, China (grant number 2021Y9160), and Fujian Medical University high-level talents scientific research start-up funding project (grant number XRCZX2017025).

1. Construction of the CRISPR/Cas9 gene knockout vectors
<ol>
	<li>Select the target genomic DNA sequence on the human CENP-E gene (GenBank Accession No. NM_001286734.2) and design the sgRNA using an online CRISPR design tool (http://crispor.tefor.net/).</li>
	<li>Input a single genomic sequence, select the genome of &#34;Homo sapiens-human-UCSC Dec 2013 (hg38 analysis set) + single nucleotide polymorphisms (SNPs): dbSNP148&#34;, and select the protospacer adjacent motif &#34;20 bp-NGG-spCas9&#34;. Choose two guide sequences, including sgRNA-1, 5&#39;-CGGCCGCACTCGCACGCAGA-3&#39; and sgRNA-2 5&#39;-TTCTTTAGAGACGCGGGCTC-3&#39;, according to the specificity scores50, the predicted efficiency10, and the minimal off-targets.</li>
	<li>Order and synthesize the single-stranded DNA oligonucleotides (ssODNs). Dilute the ssODNs in ddH2O to a final concentration of 100 &#181;M. To phosphorylate and anneal the sgRNA oligos, add 1 &#181;L of forward oligo, 1 &#181;L of reverse oligo, 0.5 &#181;L of T4 polynucleotide kinase, 1 &#181;L of T4 polynucleotide kinase buffer, and 6.5 &#181;L of RNase-free water in a polymerase chain reaction (PCR) tube, and incubate the tube at 37 &#176;C for 30 min, 95 &#176;C for 5 min; then, ramp down to 25 &#176;C at 5 &#176;C min-1.</li>
	<li>Digest the pX458 plasmid using the Bbs<img alt="Equation 1" src="/files/ftp_upload/65476/65476eq01.jpg" style="margin: auto;" /> restriction enzyme. Add 1 &#181;g of the pX458 plasmid, 2 &#181;L of 10x buffer G, 1 &#181;L of Bbs<img alt="Equation 1" src="/files/ftp_upload/65476/65476eq01.jpg" style="margin: auto;" />, and add RNase-free ddH2O to 20 &#181;L in a 1.5 mL centrifuge tube. Incubate the tube at 37 &#176;C for 2 h. Then, purify the linear plasmid using the column DNA gel extraction kit according to the manufacturer&#39;s protocols.</li>
	<li>Ligate the sgRNA oligos into the pX458 plasmid (pSpCas9(BB)-2A-green fluorescent protein (GFP) plasmid, Addgene ID. 48138). Add 1 &#181;L of the annealed oligonucleotides, 25 ng of the linear plasmid, 1 &#181;L of 10x T4 DNA ligation buffer, 0.5 &#181;L of T4 DNA ligase (350 U/&#181;L) and make up to 10 &#181;L with ddH2O in a 1.5 mL centrifuge tube. Incubate the ligation solution at 16 &#176;C for 2 h.</li>
	<li>Incubate 10 &#181;L of the constructed plasmid with the competent DH5&#945; cells on ice for 30 min, heat shock at 42 &#176;C for 45 s, and immediately place them on ice for 5 min. Add 500 &#181;L of Luria-Bertani (LB) medium and seed the cells on an LB plate containing ampicillin (100 &#181;g/mL). Incubate it at 37 &#176;C for 16 h and screen the resistant clones.</li>
	<li>Select 5-10 single colonies with a sterilized pipette tip and transfer them to a 1 mL culture of LB medium with ampicillin (100 &#181;g/mL). Incubate the culture at 37 &#176;C and shake it at 180 rpm for 12-16 h.</li>
	<li>Isolate the plasmid DNA using a plasmid extraction kit according to the manufacturer&#39;s protocols. Validate the plasmid DNA of each colony by Sanger sequencing from the U6 promoter site using the U6-Fwd primer 5&#39;-GAGGGCCTATTTCCCATGATTCC-3&#39;.</li>
	<li>Extract and purify the plasmids using the endo-free plasmid DNA kit according to the manufacturer&#39;s protocols.</li>
</ol>
2. Transfection, isolation, and screening of the CENP-E knockout HeLa cells
<ol>
	<li>Culture HeLa cells in Dulbecco Modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>Incubate the HeLa cells using 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA) at 37 &#176;C for 2-3 min, dissociate the cells by pipetting gently, and then, seed the cells in new plates at a dilution ratio of 1:4 for cell passage.</li>
	<li>Seed the cells in a 12-well plate, and culture to 60-70% confluency before transfection. Transfect the pX458-sgRNA plasmids into HeLa cells using the referenced reagents according to the manufacturer&#39;s protocols (see the Table of Materials).
	<ol>
		<li>Gently mix 1 &#181;g of validated pX458-sgRNA plasmid and 50 &#181;L of reduced serum medium in tube A. Then, gently mix 2 &#181;L of the transfection reagent and 50 &#181;L of reduced serum medium in tube B. Incubate tubes A and B separately at room temperature for 5 min.</li>
		<li>Mix both tube A and tube B gently and incubate at room temperature for 5 min. Add the mixture to a 12-well plate, incubate the cells for 6 h, and replace the medium with a fresh DMEM medium.</li>
	</ol>
	</li>
	<li>Examine HeLa cells after 24 h or 48 h using a fluorescence microscope for transfection efficiency. After transfection for 48 h, completely dissociate the transfected HeLa cells using 0.25% trypsin/EDTA at 37 &#176;C for 3-5 min, count the number of cells using a Neubauer chamber or an automated cell counter, and plate the cells into three separate 96-well plates for each transfected population according to serial dilution methods10.</li>
	<li>Return the cells to a humidified incubator and then culture them for another 1-2 weeks. Change the culture medium once every 3 days. For the phenotype-based screening and validation of CENP-E knockout, dissociate and expand the cells in 24-well or 12-well plates for 5-7 days.</li>
	<li>Screen the mutant cells with smaller cell colony diameters using an inverted microscope equipped with 10x and 20x objective lenses. 
	NOTE: CENP-E mutations usually result in a significant increase in the number of dividing cells in cell colonies, which can be one of the key indicators for screening the CENP-E mutant cells.</li>
	<li>Harvest the cells for DNA extraction using the column animal genomic DNA extraction kit according to the manufacturer&#39;s protocols. Set up the PCR reactions as follows: 0.25 &#181;L of Taq polymerase, 5 &#181;L of 10x buffer (Mg2+ plus), 4 &#181;L of dNTP mixtures, 500 ng of DNA template, 1 &#181;L of forward oligo, 1 &#181;L of reverse oligo, and adjust ddH2O to 50 &#181;L. Use the following PCR program settings for gene amplification: 98 &#176;C, 10 s; 98 &#176;C, 10 s, 55 &#176;C, 30 s, 72 &#176;C, 60 s for 33 cycles; 72 &#176;C, 10 min, and then hold at 4 &#176;C. 
	NOTE: The specific primers for cloning of the target locus are listed as follows: CENP-E target F1, 5&#39;-GAGGGTCCTGGCCATTTTCCTG-3&#39;; CENP-E target R1, 5&#39;-AGATCTCCGATCCTCCCCTGTC-3&#39;; CENP-E target F2, 5&#39;-TGGTAACTGCATTTTGGTGTTCTAC-3&#39;; CENP-E target R2, 5&#39;-CCTGTTGCAACGTGAGGGAAG-3&#39;.</li>
	<li>Ligate the target DNA into the pMD18-T vector according to the manufacturer&#39;s protocols. Transfect the ligated plasmid into competent DH5&#945; cells and culture for selection of clones.</li>
	<li>Perform Sanger sequencing to determine the types of CENP-E knockout. Isolate the plasmid DNA using a plasmid extraction kit according to the manufacturer&#39;s protocols. Carry out Sanger sequencing of the plasmid DNA of each colony using the M13 forward and reverse primers. M13F primer, 5&#39;-TGTAAAACGACGGCCAGT-3&#39;; M13R primer, 5&#39;-CAGGAAACAGCTATGACC-3&#39;.</li>
	<li>Seed the wild-type and CENP-E mutant HeLa cells on 12 mm glass coverslips in a 24-well plate, respectively. Remove the complete DMEM medium and fix the cells in 4% paraformaldehyde/phosphate-buffered saline (PBS) fixative solution at room temperature for 10 min.</li>
	<li>Stain the nuclei with the 4&#39;,6-diamidino-2-phenylindole (DAPI) solution at room temperature for 5 min. Screen and validate the CENP-E mutant cells based on the phenotype of chromosome alignment using a fluorescence microscope equipped with a Plan Fluor 40x/ Numerical Aperture (NA) 0.75 objective.</li>
	<li>Screen and validate the CENP-E knockout cells using immunofluorescence staining and analysis (see section 3) of CENP-E proteins.</li>
</ol>
3. Immunofluorescence staining and high-resolution confocal microscopy
<ol>
	<li>Fix the cells with 4% paraformaldehyde/PBS fixative solution at room temperature for 10 min and immerse for 2 x 5 min in 1x PBS. 
	NOTE: Ensure that the cells are in healthy condition and cells are collected and fixed gently to avoid metaphase cell detachment.</li>
	<li>Permeabilize the cells with 0.25% Triton X-100/PBS at room temperature for 10 min and immerse them in 1x PBS for 2 x 5 min.</li>
	<li>Block the cells with 1% BSA/PBST (0.1% Tween 20 in PBS) at room temperature for 1 h. Dilute the primary antibodies with 1% BSA/PBST and incubate the samples at 4 &#176;C for 12 h.</li>
	<li>Discard the primary antibody solutions, rinse the cells 3 x 5 min with 1x PBS, and incubate the cells with diluted secondary antibodies at room temperature for 2 h.</li>
	<li>Discard the secondary antibodies and rinse the cells in 1x PBS for 3 x 5 min.</li>
	<li>Stain the nuclei with DAPI at room temperature for 5 min. Mount the slides with the mounting medium. Seal the slides with nail polish.</li>
	<li>Observe and record the fluorescence images using a scanning confocal microscope equipped with a 63x/NA 1.40 objective.</li>
</ol>
4. Chromosome preparation and karyotype analysis
<ol>
	<li>Culture the wild-type and CENP-E-/- HeLa cells in complete DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 24 h and then, incubate the wild-type and CENP-E-/- HeLa cells with 300 nM colchicine for 5 h.</li>
	<li>Incubate the cells with 0.25% trypsin/EDTA at 37 &#176;C for 3 min and collect the cells into 1.5 mL centrifuge tubes.</li>
	<li>Centrifuge the cells at 1,000 &#215; g for 5 min at room temperature, discard the supernatants, add 1.2 mL of 0.075 mol/L KCl solution, and incubate at 37 &#176;C for 20 min.</li>
	<li>Centrifuge the cells at 1,000 &#215; g for 5 min at room temperature, discard the supernatants, add 0.2 mL of the fixative solution (methanol: glacial acetic acid = 3:1) for prefixation, and mix gently for 1 min.</li>
	<li>Centrifuge the cells at 1,000 &#215; g for 5 min at room temperature, collect the pellets, add 1.5 mL of the fixative solution at room temperature for 10 min, and then centrifuge at 1,000 &#215; g for 5 min at room temperature.</li>
	<li>Discard the supernatants, add 0.6 mL of the fixative solutions, and prepare a cell suspension. Add 3-5 drops of the cell suspensions from a height of 35-40 cm on the ice slides. 
	NOTE: Keep the height for releasing the cell suspensions onto the slides at 35-40 cm; otherwise, the chromosomes may be too dispersed or not separated from each other.</li>
	<li>Dry the slides immediately with an alcohol lamp, stain the samples with 10% Giemsa staining solution for 7 min, rinse the slides with running water for 2 min, and observe the samples using a light microscope equipped with a Plan Fluor 40x/NA 0.75 objective.</li>
</ol>
5. Cell colony formation assay
<ol>
	<li>Prepare the cell suspensions in the 6-well plates at a cell density of 1,000-2,000 cells/well. Gently shake the plates to evenly distribute the cells.</li>
	<li>Place the 6-well plates into a CO2 incubator and incubate for 2-3 weeks at 37 &#176;C with 5% CO2. Change culture medium once every 5 days. Record the images using an inverted microscope equipped with 10x and 20x objective lenses. Harvest the cells when colonies are visible (hundreds of cells within a clone).</li>
	<li>To study the effects of GSK923295, culture the cells in the 6-well plates for 24 h, and add 2 mL of GSK923295 at the final concentrations of 10, 25, 50, 100, and 200 nM. 
	NOTE: Do not move the culture dish at the early stage of clone formation to avoid cell shedding, which can form new clones and affect the screening of transfected cells.</li>
	<li>Discard the culture medium and fix the cells with 1% PFA at room temperature for 10 min. Stain the colonies with 0.1% crystal violet staining solution at room temperature for 15 min. Rinse the samples with 1x PBS three times. Record the images of cell colonies and quantify the diameters of each colony using ImageJ software. Choose the Straight line selection tool, click Analyze, choose Measure, and record the length.</li>
	<li>Rinse the colonies with 1 mL of 10% acetic acid solution for 5 min. Transfer 200 &#181;L of the supernatant solutions in a 96-well plate, and measure absorbance values using a microplate spectrophotometer at A600 nm.</li>
</ol>
6. Cell viability assay
<ol>
	<li>Seed the cells in the 24-well plates and culture the cells for 48 h to 80-90% density.</li>
	<li>Incubate the cells with 100 &#181;L of 0.25% trypsin/EDTA at 37 &#176;C for 3 min.</li>
	<li>Mix 400 &#181;L of PBS with the cells with a pipette gun. Transfer 100 &#181;L of the cell suspensions into the 96-well plate.</li>
	<li>Add 20 &#181;L of MTS solution in each well with a final concentration of 317 &#181;g/mL and mix gently according to the manufacturer&#39;s protocols. Incubate the samples at 37 &#176;C for 1-3 h in a CO2 incubator.</li>
	<li>Record the absorbance values of each well at the absorbance peak of A490 nm using a microplate spectrophotometer. Use a reference wavelength of A630 nm to subtract the background contributed by cell debris and nonspecific absorbance (Cell viability = A490nm- A630 nm).</li>
</ol>

Centromere-Associated Protein-E Gene Editing Using the CRISPR/Cas9 System

The authors have no conflicts of interest to disclose.

Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint during cell division17,19,20. Genetic deletion of CENP-E usually results in the activation of spindle assembly checkpoint, cell cycle arrest, and cell death27,29,51,52. Thus, the construction of stable CENP-...

Engineered genome editing mediates the targeted modifications of genes in a variety of cells and organisms. In eukaryotes, site-specific mutagenesis can be introduced by the applications of sequence-specific nucleases that stimulate homologous recombination of target DNA1. In recent years, several genome editing technologies, including zinc finger nucleases (ZFNs)2,3, transcription activator-like effector nucleases (TALENs)4,5, and homing meganucleases6,7, have been engineered to cleave genomes at specific sites, but these approaches require complex protein engineering and redundant experimental procedures. Studies have shown that the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an efficient gene editing technology, which specifically mediates RNA-guided, site-specific DNA cleavage in a wide variety of cells and species8,9,10,11. The CRISPR/Cas9 gene knockout technology has revolutionized the fields of basic biology, biotechnology, and medicine12.
Bacteria and most archaea have evolved an RNA-based adaptive immune system that uses CRISPR and Cas proteins to identify and destroy viruses and plasmids13. Streptococcus pyogenes Cas9 (SpCas9) endonuclease contains the RuvC-like Holliday junction resolvase (RuvC) and His-Asn-His (HNH) domain, which can efficiently mediate sequence-specific, double-stranded breaks (DSBs) by providing a synthetic single-guide RNA (sgRNA) containing CRISPR RNAs (crRNA) and trans-activating crRNA (tracrRNA)14,15,16. DSBs can be repaired through the indel-forming non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, which introduces multiple mutations, including insertions, deletions, or scar-less single nucleotide substitutions, in mammalian cells1,8. Both the error-prone NHEJ and the high-fidelity HDR pathway can be used to mediate gene knockout through insertions or deletions, which can cause frameshift mutations and premature stop codons10.
Kinesin-7 CENP-E is required for kinetochore-microtubule attachment and chromosome alignment during cell division17,18,19. Antibody microinjection20,21, siRNA depletion22,23, chemical inhibition24,25,26, and genetic deletion27,28,29 of CENP-E leads to chromosome misalignment, the activation of spindle assembly checkpoint and mitotic defects, which results in aneuploidy and chromosomal instability19,30. In mice, CENP-E deletion results in abnormal development and embryonic lethality at the very early stages of development27,29,31. Genetic deletion of CENP-E usually leads to chromosome misalignment and cell death26,27,29, which is an obstacle in studying the functions and mechanisms of the CENP-E proteins.
A recent study has established a conditional CENP-E knockout cell line using an Auxin-inducible CRISPR/Cas9 gene-editing method32, which enables rapid degradation of CENP-E proteins in a relatively short time33. However, to date, stable CENP-E knockout cell lines have not been established, which is an unresolved technical challenge in CENP-E biology. Considering genetic robustness34, genetic compensation responses35,36,37, and complex intracellular environments, as the direct consequences of complete deletion of CENP-E may be complex and unpredictable, it is important to establish CENP-E knockout cell lines for the investigation of mechanisms of chromosome alignment, spindle assembly checkpoint, and downstream signaling pathways.
The discovery and applications of CENP-E inhibitors are important for cancer treatment. To date, seven types of CENP-E inhibitors have been found and synthesized, including GSK923295 and its derivatives24,25, PF-277138,39, imidazo[1,2-a]pyridine scaffold derivatives40,41, compound-A42,43, syntelin44,45, UA6278446, and benzo[d]pyrrolo[2,1-b]thiazole derivatives47. Among these inhibitors, GSK923295 is an allosteric and efficient CENP-E inhibitor that binds to the motor domain of CENP-E and inhibits CENP-E microtubule-stimulated ATPase activity with a Ki of 3.2 &#177; 0.2 nM24,25. However, compared with the inhibitory effects of GSK923295 on cultured cancer cells, the therapeutic effects of GSK923295 in clinical cancer patients are not ideal48,49, which also raised concerns about the specificity of GSK923295 for CENP-E. Moreover, the specificity and side effects of other CENP-E inhibitors on the CENP-E proteins are key issues in cancer research.
In this study, we have completely knocked out the CENP-E gene in HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies have been established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, to improve the screening efficiency and success rate of CENP-E gene editing. Furthermore, CENP-E knockout cell lines can be used to test the specificity of candidate compounds for CENP-E.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>4S Gelred, 10,000x in water</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A616697</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10009164</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10000218</td>
			<td>Dissolve in H2O to prepare a 10% working solution.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A620014</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0423</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L)</td>
			<td>Beyotime</td>
			<td>A0460</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>100092690</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BubR1 rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab254326</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution&#160;</td>
		</tr>
		<tr>
			<td>Anti-CENP-B mouse monoclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376392</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution.</td>
		</tr>
		<tr>
			<td>Anti-CENP-E rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab133583</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Anti-fade mounting medium</td>
			<td>Beyotime</td>
			<td>P0131</td>
			<td>Slowing down the quenching of fluorescent signals.</td>
		</tr>
		<tr>
			<td>Anti-&#945;-tubulin mouse monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td>For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Biotek Epoch Microplate Spectrophotometer</td>
			<td>Biotek Instruments</td>
			<td>Biotek Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>69003435</td>
			<td></td>
		</tr>
		<tr>
			<td>BpiI (BbsI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER1011</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter 96 aqueous one solution cell proliferation assay</td>
			<td>Promega</td>
			<td>G3580</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424BK745380</td>
			<td></td>
		</tr>
		<tr>
			<td>Colchicine</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>61001563</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal scanning microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>CITOTEST</td>
			<td>80344-1220</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B528413</td>
			<td></td>
		</tr>
		<tr>
			<td>DL2000 DNA marker</td>
			<td>TaKaRa</td>
			<td>3427A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo-free plasmid mini kit <img alt="Equation 2" src="/files/ftp_upload/65476/65476eq02.jpg" style="margin: auto;" /></td>
			<td>Omega</td>
			<td>D6950</td>
			<td></td>
		</tr>
		<tr>
			<td>Ezup Column Animal Genomic DNA Purification Kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518251</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Zhejiang Tianhang Biotechnology</td>
			<td>11011-8611</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentian violet</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>71019944</td>
			<td>Dissolve in PBS to prepare 0.1% gentian violet/PBS.</td>
		</tr>
		<tr>
			<td>Giemsa staining solution</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>71020260</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 8.0 software</td>
			<td>GraphPad</td>
			<td>www.graphpad.com</td>
			<td>Statistical analysis.</td>
		</tr>
		<tr>
			<td>GSK923295</td>
			<td>MedChemExpress</td>
			<td>HY-10299</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cell line</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator</td>
			<td>Heal Force</td>
			<td>HF90/HF240</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Image processing and analysis.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nanjing Jiangnan Novel Optics</td>
			<td>XD-202</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar powder</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>A507003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipo6000 transfection reagent</td>
			<td>Beyotime</td>
			<td>C0526</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti-S2 microscope</td>
			<td>Nikon</td>
			<td>Ti-S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM&#160;reduced&#160;serum&#160;medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80096618</td>
			<td>Dissolve in PBS to prepare 4% paraformaldehyde/PBS.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>SanPrep column DNA gel extraction kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518131</td>
			<td></td>
		</tr>
		<tr>
			<td>SanPrep&#160;column&#160;plasmid&#160;mini-preps&#160;kit</td>
			<td>Sangon Biotech (Shanghai)</td>
			<td>B518191</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>TaKaRa</td>
			<td>2011A</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>TaKaRa</td>
			<td>2021A</td>
			<td></td>
		</tr>
		<tr>
			<td>TaKaRa Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30188928</td>
			<td>Dissolve in PBS to prepare 0.25% Triton X-100/PBS.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30189328</td>
			<td>Dissolve in PBS to prepare 0.1% Tween 20/PBS.</td>
		</tr>
	</tbody>
</table>

generation of centromere-associated protein-e cenp-e-/- knockout cell lines using the crispr/cas9 system

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Author Spotlight: Establishing &lt;em&gt;CENP-E&lt;/em&gt; Knockout HeLa Cells &amp;#8211; A Novel Approach to Study Kinesin-7 &lt;em&gt;CENP-E&lt;/em&gt; Biology and its Inhibitors

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.

The CENP-E-/- HeLa cells were successfully generated using the CRISPR/Cas9 system (Figure 1). The timeline and critical experimental steps of this method are shown in Figure 1. First, we designed and synthesized the CENP-E-specific sgRNAs, annealed and ligated the sgRNAs into the pX458 plasmid, transfected the plasmid into HeLa cells, and cultured them for 48 h. The transfected cells were dissociated and seeded in a 96-well plate usi...

Watch this Scientific Journal Video about Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System at JoVE.com

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

generation-centromere-associated-protein-e-cenp-e-knockout-cell-lines

Research

JoVE Journal

Biology

Transfection, Isolation, and Screening of the CENP-E Knockout HeLa Cells

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 one to four for cell passage. To transfect the plasmid into HeLa cells, mix one microgram of validated PX458 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.

Immunofluorescence Staining and High-Resolution Confocal Microscopy of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

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 63 times 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 fluorescence 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 increased 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.

Chromosome Preparation and Karyotype Analysis of CRISPR/Cas9-Mediated CENP-E Knockout HeLa Cells

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 prefix. 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's 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 40 times 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.

403 Views.  Fujian Medical University.  This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

Centromere-Associated Protein-E Gene Editing Using the CRISPR/Cas9 System

Summary