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-NG...

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

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 CENP-E Knockout HeLa Cells &#8211; A Novel Approach to Study Kinesin-7 CENP-E 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 ...