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

Édition du génome de la protéine E associée au centromère à l’aide du système CRISPR/Cas9

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.

Pleins feux sur l’auteur : Établir les cellules HeLa knock-out CENP-E - Une nouvelle approche pour étudier la biologie de la kinésine-7 CENP-E et ses inhibiteurs

Génération de lignées cellulaires knock-out Protein-E CENP-E-/- associées au centromère à l’aide du système CRISPR/Cas9

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.

Transfection, isolement et criblage des cellules HeLa knock-out CENP-E

Pour commencer, ajoutez une solution de trypsine-EDTA à 0,25 % dans les cellules HeLa et incubez à 37 degrés Celsius pendant deux à trois minutes. Dissociez doucement les cellules par pipetage et ensemencez-les dans de nouvelles plaques dans un rapport de un à quatre pour le passage des cellules. Pour transfecter le plasmide dans les cellules HeLa, mélangez un microgramme de plasmide d’ARNsg PX458 validé, avec 50 microlitres de milieu sérique réduit dans le tube A.In tube B, mélangez délicatement deux microlitres de réactif de transfection et 50 microlitres de milieu sérique réduit et incubez à température ambiante pendant cinq minutes.Ensuite, mélangez le contenu des tubes A et B et incubez à température ambiante pendant cinq minutes supplémentaires. Ajouter le mélange dans la plaque à 12 puits et incuber les cellules à 37 degrés Celsius pendant six heures. À la fin de l’incubation, remplacer le milieu par du milieu DMEM frais.Après 24 ou 48 heures, évaluez l’efficacité de la transfection en examinant les cellules HeLa au microscope à fluorescence. Dissocier complètement les cellules HeLa transfectées avec de l’EDTA à 0,25 % de trypsine et incuber à 37 degrés Celsius pendant trois à cinq minutes. Ensuite, comptez les cellules à l’aide d’une chambre Neubauer ou d’un compteur de cellules automatisé.Plaquez les cellules en trois plaques distinctes de 96 puits pour chaque population transfectée en suivant des méthodes de dilution en série. Remettez les cellules dans un incubateur humidifié pendant une à deux semaines. Pour le criblage et la validation de CENP-E Knockout basés sur le phénotype, dissocier et développer les cellules dans des plaques de 24 ou 12 puits pendant cinq à sept jours.Criblez les cellules mutantes avec des diamètres de colonie plus petits à l’aide d’un microscope inversé avec des lentilles d’objectif à grossissement 10 fois et 20 fois. Ensuite, récoltez les cellules pour l’extraction de l’ADN en utilisant le kit d’extraction d’ADN génomique animal en colonne en suivant les instructions du fabricant et configurez les réactions en chaîne par polymérase. Placez l’ADN cible dans le vecteur pMD18-T selon les instructions des protocoles du fabricant.Transfectez le plasmide ligaturé dans les cellules DH5-Alpha compétentes et procédez à la culture pour la sélection des clones. Pour identifier les types de CENP-E Knockout, isolez l’ADN plasmidique à l’aide d’un kit d’extraction de plasmide en suivant les directives du fabricant. Ensuite, effectuez le séquençage de Sanger pour l’ADN plasmidique de chaque colonie à l’aide d’amorces directes et inverses M13.Ensemencez les cellules HeLa de type sauvage et les cellules HeLa mutantes CENP-E sur des lamelles de verre de 12 millimètres dans une plaque de 24 puits. Retirez le milieu DMEM complet et fixez les cellules à l’aide d’une solution de paraformaldéhyde à 4 % et de PBS à température ambiante pendant 10 minutes. Ensuite, colorez les noyaux avec du DAPI à température ambiante pendant cinq minutes.Observer et valider les cellules mutantes CENP-E en fonction du phénotype d’alignement des chromosomes à l’aide d’un microscope à fluorescence. Le criblage phénotypique des colonies a montré que les cellules CENP-E Knockout présentaient un désalignement chromosomique accru par rapport au groupe témoin.

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 ...

Recherche

Biologie

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

Coloration par immunofluorescence et microscopie confocale haute résolution de cellules HeLa knock-out CENP-E médiées par CRISPR/Cas9

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

Préparation chromosomique et analyse du caryotype des cellules HeLa knock-out CENP-E médiées par CRISPR/Cas9