The CRISPR-Cas system derived from bacteria and archaea adaptive immune system is a high potential method for fast genome editing that promises to revolutionize previous genome engineering. It is based on the specific targeted induced double strand break by an endonuclease. In elapsed studies PA28γ figured out as an important key molecule involved in cell cycle regulation, cell signaling transcription, immune response and apoptosis. Recent investigations showed p53 to be a target of PA28γ enhanced ubiquitination via MDM2 and subsequent proteasomal degradation. Otherwise mutant p53 (R248Q) has been shown as suppressor of the REGγ promotor. This study aimed the CRISPR-Cas mediated gene knockout of PSME3 and tp53 in Caspase3 lacking MCF-7 breast cancer cells to investigate apoptosis.
A user-developed protocol was established to implement the Multiplex CRISPR/Cas9 Assembly System Kit and the alone standing pSpCas9(BB)-2A-GFP plasmid provided by Takashi Yamamoto and Feng Zhang (Ran et al. 2013, Sakuma et al. 2014) for the generation of knockout cells.
The cloning of gRNA harboring plasmids targeting PSME3 exon1/exon4 as well as tp53 exon1_1/exon1_2 was fast and in a high efficient fashion but a verification of the final constructs via T7 Endonuclease I assay was not possible.
Interestingly, using fluorescent microscopy different gRNAs cloned in the CRISPR plasmids revealed variant apparent transfection efficiencies or GFP plasmid or protein stability. Furthermore, PA28γ targeted cells showed a better survival than p53 knockout cells. Therefore, also no tp53 targeted cells survived the serial dilution and clonal selection over an eight week period. PSME3 exon1_F1, exon4_C8 and exon4_B9 revealed PA28γ levels of about 50% compared to the untransfected wild type cells in Western Blot analyses. This could be caused by a heterozygous knockout of the PSME3 gene on chromosome17. One single cell clone (PSME3 exon4_F9) maybe carrying a gain of the PSME3 gene, undergoing interchromosomal recombination or only was hidden at one allele by the Cas9 enzyme showed 75% for PA28γ levels.
In summary CRISPR-Cas enabled us probable to modify the PSME3 and tp53 gene in MCF-7 cells resulting in altered survivals of the transfected cells. Additionally, first investigations of the new established MCF-7 PSME3 knockout cell lines considering the PA28γ protein level showed a successful 50% reduction. It was not possible to study any apoptosis related behavior.
Table of Contents
1. INTRODUCTION
1.1 CRISPR-Cas9 System
1.2 The microbial origin of CRISPR-Cas9
1.2.1 Components of the adaptive immune system in streptococcus pyrogenes (s. pyrogenes)
1.2.2 Mechanism of adaptive immune response in s. pyrogenes
1.3 Experimental design for precise CRIRSPR-Cas9 mediated gene engineering
1.3.1 Guide RNA (gRNA) design
1.3.2 Construction of gRNA harboring plasmids
1.3.3 Confirmation of gRNA function
1.3.4 Advantages and limitations of CRISPR-Cas
1.4 Michigan Cancer Foundation 7 (MCF-7) breast cancer cell line
1.5 Proteasome activator 28 gamma (PA28γ)
1.6 Preliminary work and aim of the study
1.6.1 Preliminary work and background of the study
1.6.2 Aim of the study
2. MATERIAL AND METHODS
2.1 Materials
2.1.1 Laboratory equipment and instruments
2.1.2 Plastic materials and consumables
2.1.3 Chemicals, buffers and media for cell biology
2.1.4 Chemicals, buffers and enzymes for molecular biology
2.1.5 Chemicals and materials for biochemistry
2.1.6 Cell lines and bacteria
2.1.7 Antibodies
2.1.8 Kits
2.1.9 Plasmids, oligonucleotides, primer
2.1.10 Private computer software
2.1.11 Buffers and solutions
2.2 Methods
2.2.1 Cell biological methods
2.2.2 Molecular biological methods
2.2.3 Biochemical methods
2.2.4 General methods
3. RESULTS
3.1 In silico design, cloning and characterization of gRNA harboring plasmids in transfected MCF-7 cells
3.1.1 In silico analyses of genomic regions related to PSME3 exon1, exon4 and tp53 exon1_1/2 for gRNA design
3.1.2 gRNA encoding oligo nucleotides were cloned into pX330A-1x2 and pX458 plasmids
3.1.3 Apparent transfection efficiencies vary with gRNA expressing constructs
3.1.4 Different gRNA constructs reveled a various outcome in MCF-7 cells
3.2 Confirmation of gRNA constructs effects on a genomic level
3.2.1 Establishment of the genomic PCR for PSME3 and tp53 gene
3.2.2 Verification of gRNA targeting efficiencies using the T7 Endonuclease I assay
3.3 Transfection of MCF-7 cells with gRNA harboring pX330A1x2 and pX458 plasmids
3.3.1 Survival rates of single cell clones are linked to the gRNA construct
3.3.2 MCF-7 cells transfected with pX330A-1x2_PSME3exon1 and pX330A-1x2_PSME3exon4 showed a heterozygous knockout of the PSME3 gene
3.3.3 The PSME3 exon4_F9 clone revealed an anomalous PA28γ level
4. DISCUSSION
4.1 The CRSIPR-Cas9 system seems to be an innovative method for fast genome engineering but it is not that easy to use as described in literature
4.2 The transfection of MCF-7 cells with different gRNA constructs revealed variant survival
4.3 For fast generation of mutant cell lines using CRISPR-Cas9 automated monitoring is necessary
4.4 Generated knockout effects of PA28y in MCF-7 cells
5. OUTLOOK
6. SUPPLEMENT
6.1 Plasmid chart pX330A-1x2
6.2 Pasmid chart pX330S-2
6.3 Plasmid chart pX458
6.4 CRISPR-Cas transfected cell Western Blot verfiifcation
6.5 Poster Abstract VideoScan Applications in CRISPR-Cas9 Monitoring
7. REFERENCES
Research Objectives and Core Themes
This thesis investigates the application of the CRISPR-Cas9 genome editing system for the targeted knockout of the PSME3 (encoding PA28γ) and tp53 genes in the Caspase-3 deficient MCF-7 breast cancer cell line, specifically aiming to characterize the subsequent cellular survival and potential apoptotic changes.
- Functional role of PA28γ in protein degradation and apoptosis.
- Implementation of CRISPR-Cas9 cloning and transfection protocols.
- Analysis of gRNA efficiency and genomic knockout verification.
- Evaluation of phenotypic outcomes and survival in edited MCF-7 cell clones.
Excerpt from the Book
1.3.4 Advantages and limitations of CRISPR-Cas
In the last few years the CRISPR-Cas system became a very often used technique in molecular biological laboratories for genome editing because it provides several advantages compared to conventional system. It is a very fast system for editing genomic regions. Especially for creating mouse models CRISPR-Cas is capable to shorten the time from 1-2 years to 1-2 months (Young et al. 2015). Furthermore, the system can be easily adjusted to target new genomic sequences just by changing the 20nt encoding gRNA (Hsu et al. 2014) and the system is ready for multiplexing. With the pX330 plasmids it is possible to target up to seven targets with just one expression plasmid (Sakuma et al. 2014). A major advantage is that CRISPR-Cas has a very high efficiency of up to 94% (Jurkat cells) (Liang et al. 2015).
But CRISPR-Cas also has some limitations. So the targeting efficiency is closely related to the cell line or organism (Ran et al. 2013). The off-target rate is higher compared to other systems. With over 50% frequency CRISPR-Cas introduces mutation at sites other than the intended one (Zhang et al. 2015). Small genomic alteration introduced by NHEJ are difficult and expensive to identify. So it is not easy to distinguish between homozygous and heterozygous clones (Li et al. 2014).
Summary of Chapters
1. INTRODUCTION: Provides a background on genome engineering history, the microbial origins of CRISPR-Cas9, the experimental design for gRNA engineering, and the specific cell and protein models used in the study.
2. MATERIAL AND METHODS: Details the materials, equipment, cell culture conditions, transfection protocols, cloning strategies, and biochemical methods, including DNA isolation, PCR, and Western Blotting.
3. RESULTS: Presents the findings regarding gRNA design, cloning success, transfection efficiency, survival rates of cell clones, and the verification of PA28γ knockout via Western Blot analysis.
4. DISCUSSION: Analyzes the experimental success, limitations regarding transfection and genomic verification, the impact of gRNA constructs on survival, and the implications of cellular heterogeneity.
5. OUTLOOK: Suggests future directions for establishing more robust PCR and screening methods, improving clonal selection, and further investigating apoptotic pathways in PA28γ knockout cells.
6. SUPPLEMENT: Includes detailed plasmid maps, additional Western Blot validation data, and a conference abstract related to the VideoScan monitoring technology.
7. REFERENCES: Lists the cited scientific literature supporting the methodologies and theoretical frameworks applied in the study.
Keywords
CRISPR-Cas9, PA28γ, PSME3, MCF-7, Breast Cancer, Gene Knockout, Genome Engineering, Apoptosis, p53, Western Blot, Transfection, gRNA, Clonal Selection, VideoScan, Molecular Biology
Frequently Asked Questions
What is the core focus of this Master's thesis?
The thesis focuses on the functional analysis of the PA28γ protein in MCF-7 breast cancer cells by utilizing the CRISPR-Cas9 genome editing system to perform gene knockouts and evaluate the biological consequences.
Which genes were targeted for knockout?
The study specifically targeted the PSME3 gene (which encodes PA28γ) and the tp53 gene in MCF-7 cells.
What is the primary objective of this research?
The primary goal was to establish the CRISPR-Cas9 laboratory workflow and investigate the effects of targeted gene disruption on the transfected MCF-7 cell survival and apoptotic behavior.
What scientific methods were employed?
Key methods included molecular cloning of gRNA-harboring plasmids, Lipofectamine-mediated transfection, fluorescence-based clonal selection, genomic PCR, T7 Endonuclease I assays, and Western Blot analysis for protein quantification.
What are the main topics discussed in the main body?
The main body covers the in silico gRNA design, the methodology for constructing CRISPR plasmids, the transfection process, the confirmation of genomic effects, and the statistical analysis of cell survival and protein expression levels.
Which keywords best describe this research?
CRISPR-Cas9, PA28γ, MCF-7, Breast Cancer, Gene Knockout, and Genome Engineering are among the most descriptive keywords for this study.
Why was the MCF-7 cell line chosen for these experiments?
MCF-7 is a well-established breast cancer cell line that allows researchers to study the impacts of PA28γ and p53, noting specifically that these cells are known to be Caspase-3 deficient.
What were the main conclusions regarding transfection efficiency?
The research found that transfection efficiency is heavily influenced by the specific gRNA construct used, with PSME3-targeting constructs displaying lower apparent efficiencies compared to tp53-targeting ones, often correlated with cell viability issues.
What was the outcome of the PA28γ knockout?
Western Blot analyses confirmed a successful reduction in PA28γ protein levels by approximately 50%, which was interpreted as a stable heterozygous knockout in the tested clones.
- Arbeit zitieren
- Marcel Schlecht (Autor:in), 2016, Functional analysis of PA28γ in MCF-7 breast cancer cells by overexpression & CRISPR-Cas9 mediated gene silencing, München, GRIN Verlag, https://www.grin.com/document/366894
