transfection, isolation, and screening of the cenp-e knockout hela cells

Centromeer-geassocieerde eiwit-E-genbewerking met behulp van het CRISPR/Cas9-systeem

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.

Auteur in de schijnwerpers: CENP-E Knock-out HeLa-cellen vaststellen - een nieuwe benadering om kinesine-7 CENP-E-biologie en zijn remmers te bestuderen

Generatie van Centromeer-geassocieerde proteïne-E CENP-E-/- knock-outcellijnen met behulp van het CRISPR/Cas9-systeem

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.

Transperfectie, isolatie en screening van de CENP-E knock-out HeLa-cellen

Voeg om te beginnen 0,25% trypsine-EDTA-oplossing toe aan de HeLa-cellen en incubeer gedurende twee tot drie minuten bij 37 graden Celsius. Dissocieer de cellen voorzichtig door te pipetteren en zaai ze in nieuwe platen in een verhouding van één tot vier voor celpassage. Om het plasmide in HeLa-cellen te transfecteren, mengt u één microgram gevalideerd PX458 sgRNA-plasmide met 50 microliter gereduceerd serummedium in buis A.In buis B, mengt u voorzichtig twee microliter van het transfectiereagens en 50 microliter gereduceerd serummedium en incubeert u gedurende vijf minuten bij kamertemperatuur.Combineer vervolgens de inhoud van buizen A en B en incubeer nog vijf minuten bij kamertemperatuur. Voeg het mengsel toe aan de plaat met 12 putjes en incubeer de cellen gedurende zes uur bij 37 graden Celsius. Vervang aan het einde van de incubatie het medium door vers DMEM-medium.Beoordeel na 24 of 48 uur de transfectie-efficiëntie door de HeLa-cellen onder een fluorescentiemicroscoop te onderzoeken. Dissocieer de getransfecteerde HeLa-cellen volledig met 0,25% trypsine EDTA en incubeer gedurende drie tot vijf minuten bij 37 graden Celsius. Tel vervolgens de cellen met behulp van een Neubauer-kamer of een geautomatiseerde cellenteller.Plaat de cellen in drie afzonderlijke platen met 96 putjes voor elke getransfecteerde populatie volgens seriële verdunningsmethoden. Breng de cellen gedurende één tot twee weken terug naar een bevochtigde incubator. Voor fenotype-gebaseerde screening en validatie van CENP-E Knockout, dissocieert en breidt u de cellen uit in platen met 24 of 12 putjes gedurende vijf tot zeven dagen.Screen de gemuteerde cellen met kleinere koloniediameters met behulp van een omgekeerde microscoop met objectieflenzen met een vergroting van 10 keer en 20 keer. Oogst vervolgens de cellen voor DNA-extractie door gebruik te maken van de Column Animal Genomic DNA Extraction Kit volgens de instructies van de fabrikant en stel de polymerasekettingreacties in. Bind het doel-DNA in de pMD18-T-vector zoals aangegeven door de protocollen van de fabrikant.Transfecteer het geligeerde plasmide in competente DH5-Alpha-cellen en ga verder met de kweek voor kloonselectie. Om de soorten CENP-E Knockout te identificeren, isoleert u het plasmide-DNA met behulp van een plasmide-extractiekit volgens de richtlijnen van de fabrikant. Voer vervolgens Sanger-sequencing uit voor het plasmide-DNA van elke kolonie met behulp van M13 voorwaartse en achterwaartse primers.Zaai de wildtype en CENP-E mutante HeLa-cellen op 12 millimeter glazen afdekglaasjes in een plaat met 24 putjes. Verwijder het volledige DMEM-medium en fixeer de cellen met een 4%paraformaldehyde- en PBS-oplossing gedurende 10 minuten bij kamertemperatuur. Kleur vervolgens de kernen met DAPI gedurende vijf minuten bij kamertemperatuur.Observeer en valideer de CENP-E-mutante cellen op basis van het fenotype van chromosoomuitlijning met behulp van een fluorescentiemicroscoop. De fenotypische screening van de kolonies toonde aan dat de CENP-E Knockout-cellen een verhoogde chromosoomafwijking vertoonden in vergelijking met de controlegroep.

Research

JoVE Journal

Biology

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

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 ...

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

Immunofluorescentiekleuring en confocale microscopie met hoge resolutie van CRISPR/Cas9-gemedieerde CENP-E knock-out HeLa-cellen

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

Chromosoomvoorbereiding en karyotype-analyse van CRISPR/Cas9-gemedieerde CENP-E knock-out HeLa-cellen