immunofluorescence staining and high-resolution confocal microscopy of crispr/cas9-mediated cenp-e knockout hela cells

使用 CRISPR/Cas9 系统进行着丝粒相关 Protein-E 基因编辑

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.

作者聚焦：建立 CENP-E 敲除 HeLa 细胞 – 一种研究驱动蛋白 7 CENP-E 生物学及其抑制剂的新方法

使用 CRISPR/Cas9 系统生成着丝粒相关蛋白- E CENP-E-/- 敲除细胞系

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.

CRISPR/Cas9介导的 CENP-E 敲除HeLa细胞的免疫荧光染色和高分辨率共聚焦显微镜

首先，取一盘野生型和CENP-E突变HeLa细胞在盖玻片上生长。用4%多聚甲醛和PBS固定溶液在室温下固定细胞10分钟。然后，浸泡在 PBS 中两轮，每轮五分钟。接下来，在室温下用0.25%Triton X-100和PBS透化细胞10分钟。将它们浸入 PBS 中两轮，每轮五分钟。继续在室温下用 1% BSA PBS 和 0.1%Tween 20 封闭细胞一小时。在 1%BSA 和 PBST 中稀释一抗，并将样品在 4 摄氏度下孵育 12 小时。丢弃一抗溶液。然后，用PBS冲洗细胞三次，每次五分钟。向细胞中加入稀释的二抗，并在室温下孵育两小时。接下来，丢弃二抗。然后用PBS冲洗细胞三次，每次五分钟。在室温下使用DAPI染色细胞核五分钟。用安装介质安装载玻片并用指甲油密封。使用装有63倍放大倍率、1.40数值孔径物镜的扫描共聚焦显微镜观察荧光图像，并记录它们以供进一步分析。对照组和CENP-E突变组的免疫荧光染色表明，CENP-E突变克隆1细胞中CENP-E蛋白的荧光强度被完全敲除，CENP-E突变克隆3细胞中的荧光强度显著降低。与对照细胞相比，克隆 1 和 3 中染色体错位的中期细胞比例增加。同时，克隆1细胞和克隆3细胞中中期细胞的比例显著增加。此外，与对照组相比，CENP-E缺失后克隆1组和克隆3组的间期细胞比例略有增加。CENP-E缺失后前期、后期和末期细胞的发生率略有下降，而CENP-E缺失后中期细胞的发生率增加。

immunofluorescence-staining-high-resolution-confocal-microscopy

Research

JoVE Journal

Biology

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

62 次观看.  Fujian Medical University. 首先，取一盘野生型和CENP-E突变HeLa细胞在盖玻片上生长。用4%多聚甲醛和PBS固定溶液在室温下固定细胞10分钟。然后，浸泡在 PBS 中两轮，每轮五分钟。接下来，在室温下用0.25%Triton X-100和PBS透化细胞10分钟。将它们浸入 PBS 中两轮，每轮五分钟。继续在室温下用 1% BSA PBS 和 0.1%Tween 20 封闭细胞一小时。在 1%BSA 和 PBST 中稀释一抗，并将样品在 4 摄氏度下孵育 12 小时。?...

视频: CRISPR/Cas9介导的 CENP-E  敲除HeLa细胞的免疫荧光染色和高分辨率共聚焦显微镜

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 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&#160;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.

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&#160;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.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

科研

生物学

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

To begin, add 0.25%Trypsin-EDTA Solution to the HeLa cells and incubate at 37 degrees Celsius for two to three minutes. Gently dissociate the cells by pipetting and seed them in new plates at a ratio of one to four for cell passage. To transfect the plasmid into HeLa cells, mix one microgram of validated PX458 sgRNA plasmid, with 50 microliters of reduced serum medium in tube A.In tube B, gently mix two microliters of the transfection reagent and 50 microliters of reduced serum medium and incubate at room temperature for five minutes.Then, combine the contents from tubes A and B and incubate at room temperature for another five minutes. Add the mixture to the 12-well plate and incubate the cells at 37 degrees Celsius for six hours. At the end of the incubation, replace the medium with fresh DMEM medium.After 24 or 48 hours, assess the transfection efficiency by examining the HeLa cells under a fluorescence microscope. Dissociate the transfected HeLa cells fully using 0.25%trypsin EDTA and incubate at 37 degrees Celsius for three to five minutes. Next, count the cells using a Neubauer chamber or an automated cell counter.Plate the cells into three separate 96-well plates for each transfected population following serial dilution methods. Return the cells to a humidified incubator for one to two weeks. For phenotype-based screening and validation of CENP-E Knockout, dissociate and expand the cells in 24-well or 12-well plates for five to seven days.Screen the mutant cells with smaller colony diameters using an inverted microscope with 10 times and 20 times magnification objective lenses. Next, harvest the cells for DNA extraction by employing the Column Animal Genomic DNA Extraction Kit following the manufacturer's instructions and set up the polymerase chain reactions. Ligate the target DNA into the pMD18-T Vector as directed by the manufacturer's protocols.Transfect the ligated plasmid into competent DH5-Alpha cells, and proceed with the culture for clone selection. To identify the types of CENP-E Knockout, isolate the plasmid DNA with the help of a Plasmid Extraction Kit following the manufacturer's guidelines. Next, carry out Sanger sequencing for the plasmid DNA of each colony using M13 forward and reverse primers.Seed the wild type and CENP-E mutant HeLa cells on 12-millimeter glass cover slips in a 24-well plate. Remove the complete DMEM medium and fix the cells using a 4%paraformaldehyde and PBS solution at room temperature for 10 minutes. Next, stain the nuclei with DAPI at room temperature for five minutes.Observe and validate the CENP-E mutant cells based on chromosome alignment phenotype using a fluorescence microscope. The phenotypic screening of the colonies showed that the CENP-E Knockout cells showed increased chromosome misalignment compared to the control group.

CENP-E 敲除 HeLa 细胞的转染、分离和筛选

To begin, take a plate of wild type and CENP-E mutant HeLa cells growing on a cover slip. Fix the cells with 4%paraformaldehyde and PBS fixative solution at room temperature for 10 minutes. Then, immerse in PBS for two rounds of five minutes each.Next, permeabilize the cells with 0.25%Triton X-100 and PBS at room temperature for 10 minutes. Immerse them in PBS for two rounds of five minutes each. Proceed to block the cells with 1%BSA PBS with 0.1%Tween 20 at room temperature for one hour.Dilute the primary antibodies in 1%BSA and PBST and incubate the samples at four degrees Celsius for 12 hours. Discard the primary antibody solutions. Then, rinse the cells three times for five minutes each with PBS.Add diluted secondary antibodies to the cell and incubate at room temperature for two hours. Next, discard the secondary antibodies. Then rinse the cells with PBS three times for five minutes each.Stain the nuclei using DAPI at room temperature for five minutes. Mount the slides with the mounting medium and seal them with nail polish. Observe the fluorescence images using a scanning confocal microscope, fitted with a 63 times magnification, 1.40 numerical aperture objective, and record them for further analysis.Immunofluorescence staining of the control and CENP-E mutant groups showed that the fluorescence intensities of the CENP-E proteins were completely knocked out in the CENP-E mutant Clone 1 cells and significantly decreased in CENP-E mutant Clone 3 cells. The ratios of metaphase cells with chromosome misalignment increased in Clones 1 and 3 compared to the control cells. Meanwhile, the ratios of metaphase cells increased significantly in Clone 1 cells and Clone 3 cells.In addition, the ratio of interphase cells slightly increased in Clone 1 and 3 groups after CENP-E deletion, compared with the control group. The rates of prophase, anaphase, and telophase cells were slightly decreased after CENP-E deletion, whereas the rates of metaphase cells increased after CENP-E deletion.

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

To begin, take the wild type and CENP-E mutant HeLa cell cultures and add 300 nanomolar colchicine before incubating them for five hours. Next, incubate the cells with 0.25%trypsin-EDTA at 37 degrees Celsius for three minutes, and collect the cells into 1.5 milliliter centrifuge tubes. Next, centrifuge the cells at 1000 G for five minutes at room temperature, discard the supernatants and add 1.2 milliliters of 0.075 molar potassium chloride solution.Incubate the cells at 37 degrees Celsius for 20 minutes. At the end of the incubation, centrifuge the cells, discard the supernatant, and add 0.2 milliliters of fixative solution for prefix. Mix gently for one minute, then collect the cell pellets as demonstrated earlier, and add 1.5 milliliters of fixative solution at room temperature for 10 minutes.After centrifuging the cells and discarding the supernatant, add 0.6 milliliters of the fixative solutions to create a cell suspension. Carefully drop three to five droplets of the cell suspensions from 35 to 40 centimeters onto the ice slides. Immediately dry the slides using an alcohol lamp and stain them with 10%Giemsa's sustaining solution for seven minutes.Rinse the slides with running water for two minutes, then examine the samples using a light microscope equipped with a Plan Fluor 40 times magnification 0.75 numerical aperture objective. The karyotype analysis revealed decreased chromosome numbers in the CENP-E mutant HeLa cells compared with the wild type cells.