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Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System
In This Article
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-EÂ 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-EÂ 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
- 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/).
- 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
| Name | Company | Catalog Number | Comments |
| 0.25% Trypsin-EDTA | Gibco | 25200056 | |
| 1.5 mL centrifuge tube | Axygen | MCT-150-C | |
| 24-well plate | Corning | 3524 | |
| 4S Gelred, 10,000x in water | Sangon Biotech (Shanghai) | A616697 | |
| 50 mL centrifuge tube | Corning | 430828 | |
| 6 cm Petri dish | Corning | 430166 | |
| 95% ethanol | Sinopharm Chemical Reagent | 10009164 | |
| 96-well plate | Corning | 3599 | |
| Acetic acid | Sinopharm Chemical Reagent | 10000218 | Dissolve in H2O to prepare a 10% working solution. |
| Agarose | Sangon Biotech (Shanghai) | A620014 | |
| Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H+L) | Beyotime | A0428 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution. |
| Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L) | Beyotime | A0423 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution. |
| Alexa Fluor 555-labeled Donkey Anti-Mouse IgG(H+L) | Beyotime | A0460 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:500 dilution. |
| Anhydrous ethanol | Sinopharm Chemical Reagent | 100092690 | |
| Anti-BubR1 rabbit monoclonal antibody | Abcam | ab254326 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution |
| Anti-CENP-B mouse monoclonal antibody | Santa Cruz Biotechnology | sc-376392 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:50 dilution. |
| Anti-CENP-E rabbit monoclonal antibody | Abcam | ab133583 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution. |
| Anti-fade mounting medium | Beyotime | P0131 | Slowing down the quenching of fluorescent signals. |
| Anti-α-tubulin mouse monoclonal antibody | Abcam | ab7291 | For immunofluorescence. Dissolve in 1% BSA/PBST. 1:100 dilution. |
| Biotek Epoch Microplate Spectrophotometer | Biotek Instruments | Biotek Epoch | |
| Bovine Serum Albumin (BSA) | Sinopharm Chemical Reagent | 69003435 | |
| BpiI (BbsI) | Thermo Fisher Scientific | ER1011 | |
| CellTiter 96 aqueous one solution cell proliferation assay | Promega | G3580 | |
| Centrifuge | Eppendorf | 5424BK745380 | |
| Colchicine | Sinopharm Chemical Reagent | 61001563 | |
| Confocal scanning microscope | Leica | Leica TCS SP8 | |
| Coverslip | CITOTEST | 80344-1220 | |
| DAPI | Beyotime | C1006 | |
| DH5α competent cells | Sangon Biotech (Shanghai) | B528413 | |
| DL2000 DNA marker | TaKaRa | 3427A | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | C11995500BT | |
Endo-free plasmid mini kit  | Omega | D6950 | |
| Ezup Column Animal Genomic DNA Purification Kit | Sangon Biotech (Shanghai) | B518251 | |
| Fetal bovine serum | Zhejiang Tianhang Biotechnology | 11011-8611 | |
| Gentian violet | Sinopharm Chemical Reagent | 71019944 | Dissolve in PBS to prepare 0.1% gentian violet/PBS. |
| Giemsa staining solution | Sinopharm Chemical Reagent | 71020260 | |
| GraphPad Prism version 8.0 software | GraphPad | www.graphpad.com | Statistical analysis. |
| GSK923295 | MedChemExpress | HY-10299 | |
| HeLa cell line | ATCC | CCL-2 | |
| Humidified incubator | Heal Force | HF90/HF240 | |
| Image J software | National Institutes of Health | https://imagej.nih.gov/ij/ | Image processing and analysis. |
| Inverted microscope | Nanjing Jiangnan Novel Optics | XD-202 | |
| LB agar powder | Sangon Biotech (Shanghai) | A507003 | |
| Lipo6000 transfection reagent | Beyotime | C0526 | |
| Nikon Ti-S2 microscope | Nikon | Ti-S2 | |
| Opti-MEM reduced serum medium | Gibco | 31985070 | |
| Paraformaldehyde | Sinopharm Chemical Reagent | 80096618 | Dissolve in PBS to prepare 4% paraformaldehyde/PBS. |
| Penicillin-streptomycin solution | HyClone | SV30010 | |
| SanPrep column DNA gel extraction kit | Sangon Biotech (Shanghai) | B518131 | |
| SanPrep column plasmid mini-preps kit | Sangon Biotech (Shanghai) | B518191 | |
| T4 DNA ligase | TaKaRa | 2011A | |
| T4 polynucleotide kinase | TaKaRa | 2021A | |
| TaKaRa Ex Taq | TaKaRa | RR001A | |
| Triton X-100 | Sinopharm Chemical Reagent | 30188928 | Dissolve in PBS to prepare 0.25% Triton X-100/PBS. |
| Tween 20 | Sinopharm Chemical Reagent | 30189328 | Dissolve in PBS to prepare 0.1% Tween 20/PBS. |
References
- 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).
- Bibikova, M., Beumer, K., Trautman, J. K., Carroll, D.
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