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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Biology

Generering af centromer-associeret protein-E CENP-E-/- knockout-cellelinjer ved hjælp af CRISPR/Cas9-systemet

Published: June 23, 2023
doi:
10.3791/65476
, , , , , , , ,
1Department of Cell Biology and Genetics, The School of Basic Medical Sciences,Fujian Medical University, 2Key Laboratory of Stem Cell Engineering and Regenerative Medicine,Fujian Province University, 3College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics,Fujian Medical University, 4Medical Research Center,Fujian Maternity and Child Health Hospital

Summary

Denne artikel rapporterer konstruktionen af centromer-associeret protein-E (CENP-E) knockout-celler ved hjælp af CRISPR / Cas9-systemet og tre fænotypebaserede screeningsstrategier. Vi har brugt CENP-E knockout-cellelinjen til at etablere en ny tilgang til validering af CENP-E-hæmmernes specificitet og toksicitet, hvilket er nyttigt til lægemiddeludvikling og biologisk forskning.

Abstract

CRISPR (clustered regular interspaced short palindromic repeats)/Cas9-systemet har vist sig at være et kraftfuldt værktøj til præcis og effektiv genredigering i en række forskellige organismer. Centromerassocieret protein-E (CENP-E) er en plus-end-rettet kinesin, der kræves til kinetochore-mikrotubulusindfangning, kromosomjustering og spindelsamlingskontrolpunkt. Selvom cellulære funktioner af CENP-E-proteinerne er blevet godt undersøgt, har det været vanskeligt at studere de direkte funktioner af CENP-E-proteiner ved hjælp af traditionelle protokoller, fordi CENP-E-ablation normalt fører til spindelsamlingskontrolpunktaktivering, cellecyklusstop og celledød. I dette studie har vi fuldstændig slået CENP-E-genet ud i humane HeLa-celler og med succes genereret CENP-E-/- HeLa-cellerne ved hjælp af CRISPR/Cas9-systemet.

Tre optimerede fænotypebaserede screeningsstrategier blev etableret, herunder cellekoloniscreening, kromosomjusteringsfænotyper og de fluorescerende intensiteter af CENP-E-proteiner, som effektivt forbedrer screeningseffektiviteten og den eksperimentelle succesrate for CENP-E-knockout-cellerne . Det er vigtigt, at CENP-E-sletning resulterer i kromosomforskydning, den unormale placering af BUB1 mitotiske checkpoint serin / threonin kinase B (BubR1) proteiner og mitotiske defekter. Desuden har vi brugt CENP-E knockout HeLa-cellemodellen til at udvikle en identifikationsmetode for CENP-E-specifikke hæmmere.

I denne undersøgelse er der etableret en nyttig tilgang til validering af CENP-E-hæmmeres specificitet og toksicitet. Desuden præsenterer dette papir protokollerne for CENP-E-genredigering ved hjælp af CRISPR / Cas9-systemet, som kunne være et kraftfuldt værktøj til at undersøge mekanismerne for CENP-E i celledeling. Desuden vil CENP-E-knockout-cellelinjen bidrage til opdagelsen og valideringen af CENP-E-hæmmere, som har vigtige konsekvenser for udvikling af antitumorlægemidler, undersøgelser af celledelingsmekanismer i cellebiologi og kliniske anvendelser.

Introduction

Konstrueret genomredigering medierer målrettede modifikationer af gener i en række celler og organismer. I eukaryoter kan stedspecifik mutagenese indføres ved anvendelse af sekvensspecifikke nukleaser, der stimulerer homolog rekombination af mål-DNA1. I de senere år er flere genomredigeringsteknologier, herunder zinkfingernukleaser (ZFN’er)2,3, transkriptionsaktivatorlignende effektornukleaser (TALEN’er)4,5 og homing meganucleaser 6,7, blevet konstrueret til at spalte genomer på bestemte steder, men disse tilgange kræver kompleks proteinteknik og overflødige eksperimentelle procedurer. Undersøgelser har vist, at type II prokaryot grupperet regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) / Cas-systemet er en effektiv genredigeringsteknologi, der specifikt medierer RNA-styret, stedspecifik DNA-spaltning i en lang række celler og arter 8,9,10,11. CRISPR/Cas9-genets knockout-teknologi har revolutioneret områderne grundlæggende biologi, bioteknologi og medicin12.

Bakterier og de fleste arkæer har udviklet et RNA-baseret adaptivt immunsystem, der bruger CRISPR- og Cas-proteiner til at identificere og ødelægge vira og plasmider13. Streptococcus pyogenes Cas9 (SpCas9) endonuklease indeholder det RuvC-lignende Holliday-krydsresolvase (RuvC) og His-Asn-His (HNH) domæne, som effektivt kan formidle sekvensspecifikke, dobbeltstrengede pauser (DSB’er) ved at tilvejebringe et syntetisk enkeltguide-RNA (sgRNA) indeholdende CRISPR-RNA’er (crRNA) og transaktiverende crRNA (tracrRNA)14,15,16. DSB’er kan repareres gennem den indeldannende ikke-homologe endesammenføjning (NHEJ) eller homologi-rettede reparationsvej (HDR), som introducerer flere mutationer, herunder indsættelser, deletioner eller arløse enkeltnukleotidsubstitutioner, i pattedyrceller 1,8. Både den fejlbehæftede NHEJ og high-fidelity HDR-vejen kan bruges til at formidle genknockout gennem indsættelser eller deletioner, hvilket kan forårsage frameshift-mutationer og for tidlige stopkodoner10.

Kinesin-7 CENP-E er påkrævet til kinetochore-mikrotubulusbinding og kromosomjustering under celledeling 17,18,19. Antistofmikroinjektion 20,21, siRNA-udtømning22,23, kemisk hæmning 24,25,26 og genetisk deletion 27,28,29 af CENP-E fører til kromosomforskydning, aktivering af spindelsamlingskontrolpunkt og mitotiske defekter, hvilket resulterer i aneuploidi- og kromosomal ustabilitet19,30. Hos mus resulterer CENP-E-deletion i unormal udvikling og embryonal dødelighed i de meget tidlige udviklingsstadier 27,29,31. Genetisk deletion af CENP-E fører normalt til kromosomforskydning og celledød 26,27,29, hvilket er en hindring for at studere funktionerne og mekanismerne i CENP-E-proteinerne.

En nylig undersøgelse har etableret en betinget CENP-E knockout-cellelinje ved hjælp af en auxin-inducerbar CRISPR/Cas9-genredigeringsmetode32, som muliggør hurtig nedbrydning af CENP-E-proteiner på relativt kort tid33. Til dato er der imidlertid ikke etableret stabile CENP-E knockout-cellelinjer, hvilket er en uløst teknisk udfordring i CENP-E-biologien. I betragtning af genetisk robusthed34, genetiske kompensationsresponser 35,36,37 og komplekse intracellulære miljøer, da de direkte konsekvenser af fuldstændig sletning af CENP-E kan være komplekse og uforudsigelige, er det vigtigt at etablere CENP-E-knockout-cellelinjer til undersøgelse af mekanismer for kromosomjustering, spindelsamlingskontrolpunkt og nedstrøms signalveje.

Opdagelsen og anvendelsen af CENP-E-hæmmere er vigtig for kræftbehandling. Til dato er syv typer CENP-E-hæmmere blevet fundet og syntetiseret, herunder GSK923295 og dets derivater24,25, PF-277138,39, imidazo[1,2-a]pyridinstilladsderivater40,41, forbindelse-A42,43, syntelin44,45, UA6278446 og benzo[d]pyrrolo[2,1-b]thiazolderivater47. Blandt disse hæmmere er GSK923295 en allosterisk og effektiv CENP-E-hæmmer, der binder til det motoriske domæne af CENP-E og hæmmer CENP-E mikrotubuli-stimuleret ATPase-aktivitet med en Ki på 3,2 ± 0,2 nM24,25. Sammenlignet med de hæmmende virkninger af GSK923295 på dyrkede kræftceller er de terapeutiske virkninger af GSK923295 hos kliniske kræftpatienter imidlertid ikke ideelle48,49, hvilket også gav anledning til bekymring over specificiteten af GSK923295 for CENP-E. Desuden er andre CENP-E-hæmmeres specificitet og bivirkninger på CENP-E-proteinerne centrale spørgsmål i kræftforskningen.

I dette studie har vi fuldstændig slået CENP-E-genet ud i HeLa-celler ved hjælp af CRISPR/Cas9-systemet. Tre optimerede fænotypebaserede screeningsstrategier er blevet etableret, herunder cellekoloniscreening, kromosomjusteringsfænotyper og de fluorescerende intensiteter af CENP-E-proteiner, for at forbedre screeningseffektiviteten og succesraten for CENP-E-genredigering. Desuden kan CENP-E knockout-cellelinjer bruges til at teste specificiteten af kandidatforbindelser til CENP-E.

Protocol

1. Konstruktion af CRISPR/Cas9-genets knockout-vektorer Vælg målgenomisk DNA-sekvens på det humane CENP-E-gen (GenBank-tiltrædelsesnr . NM_001286734.2) og designe sgRNA’et ved hjælp af et online CRISPR-designværktøj (http://crispor.tefor.net/). Indtast en enkelt genomisk sekvens, vælg genomet af “Homo sapiens-human-UCSC Dec 2013 (hg38 analysesæt) + enkeltnukleotidpolymorfier (SNP’er): dbSNP148”, og vælg protospaceren tilstødende motiv “20 bp-NGG-spCas9”. V…

Representative Results

CENP-E-/- HeLa-cellerne blev genereret med succes ved hjælp af CRISPR/Cas9-systemet (figur 1). Tidslinjen og de kritiske eksperimentelle trin i denne metode er vist i figur 1. Først designede og syntetiserede vi de CENP-E-specifikke sgRNA’er, udglødede og ligerede sgRNA’erne i pX458-plasmidet, transfekterede plasmidet til HeLa-celler og dyrkede dem i 48 timer. De transfekterede celler blev dissocieret og podet i en 96-brøndplade …

Discussion

Kinesin-7 CENP-E er en nøgleregulator i kromosomjustering og spindelsamlingskontrolpunkt under celledeling 17,19,20. Genetisk deletion af CENP-E resulterer normalt i aktivering af spindelsamlingskontrolpunkt, cellecyklusstop og celledød 27,29,51,52. Således er konstruktionen af stabile CENP-E kn…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker alle medlemmer af cytoskeletlaboratoriet ved Fujian Medical University for nyttige diskussioner. Vi takker Jun-Jin Lin, Zhi-Hong Huang, Ling Lin, Li-Li Pang, Lin-Ying Zhou, Xi Lin og Min-Xia Wu på Public Technology Service Center, Fujian Medical University for deres tekniske assistance. Vi takker Si-Yi Zheng, Ying Lin og Qi Ke på Experimental Teaching Center of Basic Medical Sciences ved Fujian Medical University for deres støtte. Denne undersøgelse blev støttet af følgende bevillinger: National Natural Science Foundation of China (bevillingsnummer 82001608 og 82101678), Natural Science Foundation of Fujian-provinsen, Kina (bevillingsnummer 2019J05071), Joint Funds for Innovation of Science and Technology, Fujian-provinsen, Kina (bevillingsnummer 2021Y9160) og Fujian Medical University talenter videnskabeligt forskningsfinansieringsprojekt på højt niveau (bevillingsnummer XRCZX2017025).

Materials

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 Equation 2 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.
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Keywords: CENP-ECRISPR/Cas9KnockoutHeLa CellsChromosome AlignmentMitotic DefectsCell Cycle ArrestCell DeathKinetochore-microtubule CaptureSpindle Assembly Checkpoint
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Xu, M., Chen, J., Xu, Y., Zhang, J., Zhou, Y., He, J., Wu, S., Wei, Y., She, Z. Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System. J. Vis. Exp. (196), e65476, doi:10.3791/65476 (2023).
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