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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 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.

Introduction

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 engi....

Protocol

1. Construction of the CRISPR/Cas9 gene knockout vectors

  1. 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/).
  2. Input a single genomic sequence, select the genome of "Homo sapiens-human-UCSC Dec 2013 (hg38 analysis set) + single nucleotide polymorphisms (SNPs): dbSNP148", and select the protospacer adjacent motif "20 bp-NG.......

Representative Results

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.......

Discussion

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-.......

Acknowledgements

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 Fu....

Materials

NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGibco25200056
1.5 mL centrifuge tubeAxygenMCT-150-C
24-well plateCorning3524
4S Gelred, 10,000x in waterSangon Biotech (Shanghai)A616697
50 mL centrifuge tubeCorning430828
6 cm Petri dishCorning430166
95% ethanolSinopharm Chemical Reagent10009164
96-well plateCorning3599
Acetic acidSinopharm Chemical Reagent10000218Dissolve in H2O to prepare a 10% working solution.
AgaroseSangon Biotech (Shanghai)A620014
Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L)BeyotimeA0428For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L)BeyotimeA0423For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L)BeyotimeA0460For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution.
Anhydrous ethanolSinopharm Chemical Reagent100092690
Anti-BubR1 rabbit monoclonal antibodyAbcamab254326For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution 
Anti-CENP-B mouse monoclonal antibodySanta Cruz Biotechnologysc-376392For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution.
Anti-CENP-E rabbit monoclonal antibodyAbcamab133583For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Anti-fade mounting mediumBeyotimeP0131Slowing down the quenching of fluorescent signals.
Anti-α-tubulin mouse monoclonal antibodyAbcamab7291For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution.
Biotek Epoch Microplate SpectrophotometerBiotek InstrumentsBiotek Epoch
Bovine Serum Albumin (BSA)Sinopharm Chemical Reagent69003435
BpiI (BbsI)Thermo Fisher ScientificER1011
CellTiter 96 aqueous one solution cell proliferation assayPromegaG3580
CentrifugeEppendorf5424BK745380
ColchicineSinopharm Chemical Reagent61001563
Confocal scanning microscopeLeicaLeica TCS SP8
CoverslipCITOTEST80344-1220
DAPIBeyotimeC1006
DH5α competent cellsSangon Biotech (Shanghai)B528413
DL2000 DNA markerTaKaRa3427A
Dulbecco's Modified Eagle Medium (DMEM)GibcoC11995500BT
Endo-free plasmid mini kit figure-materials-4202OmegaD6950
Ezup Column Animal Genomic DNA Purification KitSangon Biotech (Shanghai)B518251
Fetal bovine serumZhejiang Tianhang Biotechnology11011-8611
Gentian violetSinopharm Chemical Reagent71019944Dissolve in PBS to prepare 0.1% gentian violet/PBS.
Giemsa staining solutionSinopharm Chemical Reagent71020260
GraphPad Prism version 8.0 softwareGraphPadwww.graphpad.comStatistical analysis.
GSK923295MedChemExpressHY-10299
HeLa cell lineATCCCCL-2
Humidified incubatorHeal ForceHF90/HF240
Image J softwareNational Institutes of Healthhttps://imagej.nih.gov/ij/Image processing and analysis.
Inverted microscopeNanjing Jiangnan Novel OpticsXD-202
LB agar powderSangon Biotech (Shanghai)A507003
Lipo6000 transfection reagentBeyotimeC0526
Nikon Ti-S2 microscopeNikonTi-S2
Opti-MEM reduced serum mediumGibco31985070
ParaformaldehydeSinopharm Chemical Reagent80096618Dissolve in PBS to prepare 4% paraformaldehyde/PBS.
Penicillin-streptomycin solutionHyCloneSV30010
SanPrep column DNA gel extraction kitSangon Biotech (Shanghai)B518131
SanPrep column plasmid mini-preps kitSangon Biotech (Shanghai)B518191
T4 DNA ligaseTaKaRa2011A
T4 polynucleotide kinaseTaKaRa2021A
TaKaRa Ex TaqTaKaRaRR001A
Triton X-100Sinopharm Chemical Reagent30188928Dissolve in PBS to prepare 0.25% Triton X-100/PBS.
Tween 20Sinopharm Chemical Reagent30189328Dissolve in PBS to prepare 0.1% Tween 20/PBS.

References

  1. Jiang, W., Bikard, D., Cox, D., Zhang, F., Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology. 31 (3), 233-239 (2013).
  2. Bibikova, M., Beumer, K., Trautman, J. K., Carroll, D.

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