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

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 meganucleases^6,7, 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-NGG-spCas9". Choose two guide sequences, including sgRNA-1, 5'-CGGCCGCACTCGCACGCAGA-3' and sgRNA-2 5'-TTCTTTAGAGACGCGGGCTC-3', according to the specificity scores

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 division^17,19,20. Genetic deletion of CENP-E usually results in the activation of spindle assembly checkpoint, cell cycle arrest, and cell death^27,29,51,52. Thus, the construction of stable CENP-.......

Materials

List of materials used in this article

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.