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

Sentromer-assosiert protein-E-genredigering ved bruk av CRISPR/Cas9-systemet

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.

Forfatter Spotlight: Etablering av CENP-E Knockout HeLa-celler – En ny tilnærming for å studere Kinesin-7 CENP-E-biologi og dens hemmere

Generering av sentromerassosiert protein-E CENP-E-/- Knockout-cellelinjer ved bruk av CRISPR/Cas9-systemet

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.

Transfeksjon, isolering og screening av CENP-E Knockout HeLa-cellene

For å begynne, tilsett 0,25% Trypsin-EDTA-løsning til HeLa-cellene og inkuber ved 37 grader Celsius i to til tre minutter. Desosiere cellene forsiktig ved å pipettere og frø dem i nye plater i forholdet en til fire for cellepassasje. For å transfektere plasmidet i HeLa-celler, bland ett mikrogram validert PX458 sgRNA-plasmid, med 50 mikroliter redusert serummedium i rør A.In rør B, bland forsiktig to mikroliter av transfeksjonsreagenset og 50 mikroliter redusert serummedium og inkuber ved romtemperatur i fem minutter.Kombiner deretter innholdet fra rør A og B og inkuber ved romtemperatur i ytterligere fem minutter. Tilsett blandingen til 12-brønnplaten og inkuber cellene ved 37 grader Celsius i seks timer. På slutten av inkubasjonen, erstatt mediet med friskt DMEM-medium.Etter 24 eller 48 timer, vurder transfeksjonseffektiviteten ved å undersøke HeLa-cellene under et fluorescensmikroskop. Dissocier de transfekterte HeLa-cellene fullt ut ved hjelp av 0,25% trypsin EDTA og inkuber ved 37 grader Celsius i tre til fem minutter. Deretter teller du cellene ved hjelp av et Neubauer-kammer eller en automatisert celleteller.Plate cellene i tre separate 96-brønnsplater for hver transfekterte populasjon etter serielle fortynningsmetoder. Returner cellene til en fuktet inkubator i en til to uker. For fenotypebasert screening og validering av CENP-E Knockout, dissosiere og utvide cellene i 24-brønns eller 12-brønnsplater i fem til syv dager.Skjerm mutantcellene med mindre kolonidiametre ved hjelp av et invertert mikroskop med 10 ganger og 20 ganger forstørrelse objektivlinser. Deretter høster du cellene for DNA-ekstraksjon ved å bruke Column Animal Genomic DNA Extraction Kit etter produsentens instruksjoner og sette opp polymerasekjedereaksjonene. Liger mål-DNA-et inn i pMD18-T-vektoren som instruert av produsentens protokoller.Transfekt det ligerte plasmidet til kompetente DH5-alfa-celler, og fortsett med kulturen for klonevalg. For å identifisere typene CENP-E Knockout, isoler plasmid-DNA ved hjelp av et plasmidekstraksjonssett i henhold til produsentens retningslinjer. Deretter utfører du Sanger-sekvensering for plasmid-DNA i hver koloni ved hjelp av M13 fremover og bakover primere.Frø av villtypen og CENP-E mutante HeLa-celler på 12 millimeter glassdeksel glir i en 24-brønnsplate. Fjern hele DMEM-mediet og fest cellene ved hjelp av en 4% paraformaldehyd- og PBS-løsning ved romtemperatur i 10 minutter. Deretter flekker du kjernene med DAPI ved romtemperatur i fem minutter.Observere og validere CENP-E muterte celler basert på kromosomjusteringsfenotype ved hjelp av et fluorescensmikroskop. Fenotypisk screening av koloniene viste at CENP-E Knockout-cellene viste økt kromosomfeilstilling sammenlignet med kontrollgruppen.

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

Immunfluorescensfarging og høyoppløselig konfokalmikroskopi av CRISPR/Cas9-medierte CENP-E Knockout HeLa-celler

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

Kromosompreparasjon og karyotypeanalyse av CRISPR/Cas9-medierte CENP-E Knockout HeLa-celler