Functional analysis of PA28γ in MCF-7 breast cancer cells by overexpression & CRISPR-Cas9 mediated gene silencing

Content

I ABSTRACT

II ZUSAMMENFASSUNG

III CONTENT

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_ PSME3 exon1 and pX330A-1x2_ PSME3 exon4 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 PA28γ 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

IV FIGURES

V TABLES

VI ABBREVIATIONS

VII ACKNOWLEDGEMENT

I Abstract

The CRISPR-Cas system derived from bacteria and archaea adaptive immune system is a high potential method for fast genome editing that promise 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.

II Zusammenfassung

Das CRISPR-Cas System repräsentiert ein sehr viel versprechendes neues System zum schnellen und einfachen editieren jedes beliebigen Genoms. Es wurde aus dem adaptiven Immunsystem von Bakterien und Archaeen adaptiert und basiert auf dem zielgerichtet, induzierten Doppelstrangbruch durch eine Endonuklease. Der Proteasom Aktivator PA28γ ist als Schlüsselmolekül in vielen zellulären Funktionen wie der Zellzyklusregulation, Gentranskription, der Immunantwort und auch der Apoptose involviert. In neuen Untersuchungen wurde PA28γ ebenfalls als Stabilisator der p53-MDM2 Bindung und somit als Ubiquitinierungsverstärker charakterisiert. Dies fördert den proteasomalen Abbau von p53. Andererseits fungiert mutiertes p53 (R248Q) als Suppressor für den PSME3 Promotor. In dieser Arbeit sollten das PSME3 und tp53 Gen mit Hilfe des CRISPR-Cas Systems in der Caspase 3 defizienten MCF-7 Zelllinie zerstört und die Zellen auf ihr apoptotisches Verhalten hin untersucht werden.

Für die genomische Auslöschung der Gene wurde das „Multiplex CRISPR/Cas9 Assembly System Kit“ und der Plasmid pSpCas9(BB)-2A-GFP von Takashi Yamamoto und Feng Zhang (Ran et al. 2013, Sakuma et al. 2014) genutzt. Das Standardprotokoll wurde entsprechend angepasst.

Das Klonieren der designten gRNAs zeigte sich als einfach und sehr zuverlässig in der Anwendung, jedoch war es nicht möglich die finalen Konstrukte unter Anwendung des T7 Endonuklease Tests zu verifizieren.

Die fluoreszenzmikroskopisch bestimmten scheinbaren Trasnfektionseffizienzen der verschiedenen Konstrukte variierten von gRNA zu gRNA möglicherwiese durch eine Plasmid- oder GFP- Instabilität. Es wurde eine bessere Überlebensrate bei den PA28γ Klonen als bei den p53 Klonen beobachtet. Dies zeigte sich auch im Sterben aller tp53 transfizierten Einzelzellklone nach der seriellen Verdünnung über einem Zeitraum von acht Wochen. Western Blot Untersuchungen der Klone PSME3 exon1_F1, exon4_C8 und exon4_B9 zeigten Proteinkonzentrationen von etwa 50% für PA28γ, was auf einen heterozygoten „knockout“ Effekt des PSME3 Gens auf Chromosom 17 schließen lässt. Der PSME3 exon4_F9 Klon zeigte eine besondere Situation mit einem nur 25%igen Verlust der zellulären PA28γ Menge. Ursächlich hierfür könnte ein vermehrtes Vorhandensein des PSME3 Genes, der Prozess der interchromosomalen Rekombination oder nur ein einzelnes betroffenes Allel sein.

Es war höchstwahrscheinlich möglich mit Hilfe von CRISPR-Cas das PSME3 und tp53 Gen in MCF-7 Zellen zu verändern, was sich in veränderten Überlebensraten der transfizierten Zellen zeigte. Weiterhin konnte in ersten Untersuchungen eine Reduzierung der PA28γ Menge um etwa 50% gezeigt werden. Jedoch war es noch nicht möglich im Umfang dieser Studie das apoptotische Verhalten der Transfektanten näher zu charakterisieren.

1. Introduction

1.1 CRISPR-Cas9 System

A new era of biology dawned in the early 1970s. 1972 Paul Berg and colleagues inserted new genetic information that was derived from the lambda phage and Galactose Operon of Escherichia coli (E.coli) into DNA of the Simian Virus 40 (SV40) for the first time. For this they:

a) linearized the circular SV40 DNA b) extended the 3’ end of one strand in a defined manner using the enzyme terminal deoxynucleotidyl transferase c) made the homodeoxypolymeric extension to the complementary strand d) annealed both strands and e) used E.coli DNA polymerase and ligase for filling the gaps and sealing the nicks (Jackson et al. 1972). The story continued to the transformation of artificial plasmids (constructed of separated plasmids in vitro) into E.coli in 1973. These plasmids were biologically full functional and shown to be replicas from both of the original DNA (Cohen et al. 1973). An enormous step towards the modern genome engineering was the usage of homologous recombination (HR) for targeting selected genes via sequence homology to the donor site. HR enabled scientists e.g. to create animal models more easy than ever before just by manipulating competent germ line stem cells via knockins or knockouts. Although the integration of exogenous oligo nucleotides is very accurate, HR stayed a pretty rare event (1 in 10⁶ -10⁹ cells) (Capecchi 1989). For high throughput applications a precise and efficient genome editing technology was needed. So far four major classes of designer nucleases were designed: meganucleases, made up of microbial moving genetic elements (Smith et al. 2006), zinc finger (ZF) nucleases, rested on eukaryotic transcription factors (Urnov et al. 2005), transcription activator-like effectors (TALEs) isolated from Xanthomonas bacteria (Christian et al. 2010) and lately the RNA-guided DNA endonuclease Cas9 derived from the type II bacterial adaptive immune system (Cong et al. 2013).

1.2 The microbial origin of CRISPR-Cas9

A long time ago no system that is comparable to the adaptive immune system in eukaryotes was known in bacteria. In the 1980s the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CIRSPR-associated (Cas) genes were initially discovered (Ishino et al. 1987). In 2007 scientists identified the CRISPR locus in Streptococcus thermophilus (S. thermophilus) as an important part of the adaptive immune system of bacteria. They proved that the integration of a genomic element taken from an infectious virus brought immunity against this bacteriophage to S. thermophilus (Barrangou et al. 2007).

1.2.1 Components of the adaptive immune system in streptococcus pyrogenes (s. pyrogenes

Up to date are known three various types of adaptive immune systems among bacteria (type I, II, III) (Makarova et al. 2011). All three types degrade invading genetic material by introducing a double stand break (Ran et al. 2013).

In 2010 and 2011 type II was identified as the most suitable system for genome engineering. It just needs the Cas9 endonuclease, a mature CRISPR RNA (crRNA) and a trans- activating CRISPR RNA (tracrRNA) (Garneau et al. 2010, Deltcheva et al. 2011). The generalized CRISPR locus of streptococcus pyrogenes (s. pyrogenes) consist of the cas genes (cas9, cas1, cas2, csn2), the tracrRNA and the crRNA array with its variable number of spacers, direct repeats and the leader (Chylinski et al. 2013) (figure 1.1)

The cas9 gene encodes the Cas9 endonuclease enzyme which mediates the double strand break and also the integration of new spacers (Heler et al. 2015, Wei et al. 2015). Further nucleases are Cas1 and Cas2 (Beloglazova et al. 2008). They form hetero complexes consisting of one Cas1 homodimer and one Cas2 homodimer. The complex formation is suggested to be necessary for integration of new spacers to the locus (Nunez et al. 2014). Other elements involved in the spacer acquisition are Csn2 and the tracrRNA (Barrangou et al. 2007). Furthermore, the tracrRNA stabilizes the Cas9 enzyme during the degradation process. Spacers are specific DNA fragments derived from phages and the direct repeats are palindromic to avoid a self-targeting of the CRISPR locus (Marraffini and Sontheimer 2010, Heler et al. 2015).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.1 Canonical generalized CRISPR locus of s. pyrogenes

The CRISPR locus encodes the noncoding tracrRNA, the cas -genes and the spacers separated by the direct repeats (Hsu et al. 2014)

Graphic by Marcel Schlecht

1.2.2 Mechanism of adaptive immune response in s. pyrogenes

The CRISPR-Cas mediated immune response can be divided up into three stages: 1) spacer acquisition, 2) expression of CRISPR-Cas, 3) interference with target DNA (Marraffini and Sontheimer 2010, Wiedenheft et al. 2012, van der Oost et al. 2014) (figure 1.2).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.2 Mechanism of microbial CRISPR System type II in adaptive immunity

1) A phage infection triggers the bacterium to express the cas -genes and tracrRNA. Now different nucleases cut out or copy (it is not understood yet) a short specific virus DNA sequence upstream of a PAM sequence. This genetic material is used for new spacer material that will be integrated into the CRISPR array. During this process also a new direct repeat is created to flank the induced spacer. 2) During an infection the pre-crRNA is expressed, tracrRNA hybridize to the direct repeats and it is processed by host RNase III into short units afterwards. 3) Bound to the Cas9 endonuclease the crRNA-tracrRNA complex targets the Cas9 to the corresponding protospacer element. Cas9 recognizes the PAM sequence and degrade the invading genomic material by performing a double strand break (Hsu et al. 2014).

Graphic by Marcel Schlecht

1) Acquisition

If s. pyrogenes is infected by a phage two different scenarios of spacer acquisition can be initiated. If the virus is unknown to the bacterium; the naïve, and when there is already a spacer for the invader in the CRISPR locus; the primed (Fineran and Charpentier 2012). During stage one a short protospacer sequence from the invading DNA is inserted into the CRISPR locus as a new spacer (Heler et al. 2014). Up to date it is not known whether the spacer sequence is cut out or copied. Maybe the production of new spacers is involved in another defense system like the restriction-modification system (Dupuis et al. 2013)

The whole process is poorly understood but like described in 1.2.1, a complex formation of Cas1 and Cas2 seems to be necessary for protospacer selection and generation of new spacer material followed by insertion into the CRISPR locus. It is suggested, that Cas2 activity is expandable but nevertheless the enzyme is important because Cas1 binds DNA in a Cas2 dependent manner (Nunez et al. 2014). Additionally involved elements during stage one are Cas9, Csn2 and tracrRNA as described before (1.2.1).

For the spacer selection another certain element is required. Sequence analysis of target DNA has shown a three nucleotides motif downstream of the target sequence, named protospacer adjacent motif (PAM). For s. pyrogenes it is NGG. PAM sequences are not included in the direct repeats of the crRNA array. So a self-targeting and self-cleavage is nearly impossible (Marraffini and Sontheimer 2010). During spacer acquisition the Cas9 enzyme identifies the PAM in the virus genome as a mutation. So only the protospacer sequence becomes new spacer material (Heler et al. 2015).

2) Expression of CRISPR-Cas

During Expression also called crRNA biogenesis, the CRISPR locus is transcribed and a CRISPR ribonucleoprotein (crRNP) complex is formed. Important for the transcription start is the leader region on the crRNA array (Pougach et al. 2010, Pul et al. 2010). It includes promotor elements, elements necessary for spacer integration as well as regulatory protein binding sites (Carte et al. 2014). After transcription the long pre-crRNA base pairs with tracrRNA at its direct repeats and it is processed by host RNase III into short units. Also Cas9 is involved in the processing but the role is nearly unclear (Deltcheva et al. 2011, Jinek et al. 2012). These short units are made up of a spacer and a direct repeat bound a tracrRNA. They are called crRNA-tracrRNA complexes. In the end the crRNA is 5’ trimmed by an unknown nuclease. During this the crRNA-tracrRNA complex is bound to the Cas9 (Jinek et al. 2012).

3) Interference with target DNA

The interference is the last step of the CRISPR immune response system type II. Here the crRNA-tracrRNA Cas9 complex translocates to the corresponding protospacer element in the phage genome and trigger degradation (Brouns et al. 2008, Garneau et al. 2010). For target recognition and degradation only Cas9 enzyme bound to crRNA and tracrRNA are required (Deltcheva et al. 2011). The structural analysis of Cas9 in complex with crRNA- tracrRNA by X-ray crystallography identified widely parts of the mechanism behind the interference (figure 1.3) (Nishimasu et al. 2014). So different lobes mediating the target recognition and nuclease activity were discovered. The recognition lobe bind both the target DNA and the crRNA-tracrRNA complex in a positively charged grove. Furthermore, two nuclease domains were revealed, entitled HNH and RuvC. These domains cleave the plus and minus strand of the target, respectively (Nishimasu et al. 2014). A key step in the activation process of Cas9 endonuclease is the loading of the crRNA, because thereby a reorientation of structural lobes inside the enzyme is performed that facilitate the DNA substrate binding (Jinek et al. 2014).

illustration not visible in this excerpt

Figure 1.3 Crystal structure of s. pyrogenes Cas9 bound to target and guide RNA Modified according to Nishimasu et al. 2014.

1.3 Experimental design for precise CRIRSPR-Cas9 mediated gene engineering

Genetic editing using the designer nuclease Cas9 introduces a double-strand break (DSB) at a specific target locus (Hsu and Zhang 2012). After cleavage by the Cas9 enzyme the cell undergoes one of two major DNA repair pathways. The nonhomologous end joining (NHEJ) or the homology-direct repair (HDR) pathway. Both pathways can be used by human to achieve a desired genomic alteration. For introducing an insertion/deletion (indel) mutation the absence of an artificial repair template is required. So the cell will undergo NHEJ which might result in a frameshift in a coding exon with premature stop codons promoting a gene knockout (Perez et al. 2008).

The alternative is the HDR pathway. HDR is able to mediate precise, specific genomic modification. For this an exogenously co-transfected repair template is necessary. It doesn’t matter if the repair template is conventional double-stranded or single-stranded. But the repair template needs homological arms to the target flanking the insertion sequence. It is possible to make small alterations in the genome like single nucleotide mutations (Chen et al. 2011). But a limitation is provided by HDRs only activity in dividing cells. Furthermore the efficiency depends close to the cell type, the repair template and the genomic locus (Saleh-Gohari and Helleday 2004).

1.3.1 Guide RNA (gRNA) design

As in the bacterial origin the 20nt guide sequence (in bacteria called spacer) determines the specificity of the Cas9. Also in the in vitro system a PAM sequence is required immediately downstream of the 20nt gRNA sequence at the genomic target. The PAM sequence must not be a part of the gRNA sequence to avoid a self-targeting and the Cas9 mediated DSB will be 3nt upstream of the PAM sequence (Ran et al. 2013).

In the selection of a gRNA there are two main considerations: 5’ of the 20nt sequence must be a PAM sequence for s. pyrogenes and the off-target effects should be as small as possible. To achieve a low off-target activity it is recommended to use an online CRISPR design tool. Such online tools calculated the off-target effects based on the base-pairing mismatch identity, position and distribution and give out a ranking of possible gRNAs for the given target sequence (Ran et al. 2013).

Because the U6 RNA polymerase III promotor is used for gRNA expression the first base 5’ of the 20nt sequence should be a guanine. The U6 promotor prefers guanines as first nucleotide of the transcript (Guschin et al. 2010).

1.3.2 Construction of gRNA harboring plasmids

There are two different possibilities for delivering gRNAs: as PCR amplicons containing an expression cassette or as gRNA expressing plasmids (Ran et al. 2013). Because the PCR based variant was not used in this study it is not explained more in detail. There is a wide variety of CRISPR plasmids for several applications. Most common are plasmids with the pX-backbone. These expression plasmids encode the Cas9 endonuclease, the tracrRNA and a cloning scaffold for the 20nt gRNA. Furthermore, some vectors are harboring a 2A-GFP or 2A-Puro fused to the Cas9 for a facilitated screening in eukaryotes (Ran et al. 2013). Other plasmids allow the simultaneous expression of up to 7 gRNA (Sakuma et al. 2014).

The plasmid construction is a fast and easy one or two step cloning procedure (depending on the plasmid) (Ran et al. 2013, Sakuma et al. 2014). Required are only two complementary single-strand oligonucleotides (gRNA) which are needed to be annealed, hybridized and ligated into the digested plasmid (Ran et al. 2013). The plasmid digestion is mediated by special restriction enzymes e.g. BpiI and Eco31I. These enzymes cut out their own recognition sequence during the digestion. So the digestion and ligation reaction can be performed in a one tube reaction at the same time without a loss in efficiency (Sakuma et al. 2014). After cloning and plasmid purification from bacteria the constructs need to be sequenced.

1.3.3 Confirmation of gRNA function

To verify the function, gRNA constructs quality must tested e.g. by T7 Endonuclease I digestion or sequencing before they are used for genome editing experiments. For both specific primer pairs are necessary to amplify the targeted region (Ran et al. 2013). The T7 Endonuclease I assay is a widely used method to detect non-perfect matched DNA after genome editing experiments. The T7 Endonuclease I is an enzyme that is capable to detect these non-perfect matches and cleave the DNA at these points (Guschin et al. 2010). The assay was described first in 2010. In preparation wild type cells are transfected with the gRNA harboring constructs and after a few days of cultivation the genomic DNA is isolated out of the harvested cell pools (Guschin et al. 2010). For the PCR amplification of the desired regions specific primers are needed. It is important that the primer binding sites have nearly an equal distance to the targeted region and that the resulting PCR product is about 400-800bp in length to allow a good visualization later (Ran et al. 2013). The PCR products must get denaturated and reannealed. So a mixture of three various reannealing products is created: two original strands without an alteration, two edited strands and a hybrid consisting of an original and an altered strand. Then the T7 Endonuclease I is enabled to recognize the hybrid product and cut the double strand about 3nt downstream of the first non-perfectly matched base pair. The digestion can be analyzed via agarose gel electrophoresis or capillary electrophoresis. Capillary electrophoresis allows a quantification of the targeting efficiency of gRNA constructs (Guschin et al. 2010). The procedure is displayed in figure 1.4.

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.4 T7 Endonuclease I assay

Eukaryotic cells get transfected with gRNA harboring plasmids. The genomic DNA is isolated from the pools and the edited loci are amplified via PCR. All PCR products are denaturated and hybridized. Possible reannealing products are two original strands, two altered strands or a hybrid of an original and an altered strand. T7Endonuclease I binds to non-perfectly matched DNA sequences and cleave the double strand. The digestion is investigated by agarose gel electrophoresis or for assuming a quantification of the gRNA target efficiency via capillary electrophoresis (Guschin et al. 2010).

Graphic by Marcel Schlecht

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).

1.4 Michigan Cancer Foundation 7 (MCF-7) breast cancer cell line

Cancer is one of the leading health problems worldwide and the second frequent reason for death. In 2015 breast cancer was the commonest new diagnosed cancer and the second most common cause for cancer death in the United States of America (Siegel et al. 2015). MCF-7 cell line was derived from a 69-year old Caucasian woman in 1970 and established by Herbert Soule (Soule et al. 1973). It was the first stable breast cancer cell line worldwide and also the source of much knowledge about mammary carcinomas (Levenson and Jordan 1997). MCF-7 cells express the cytoplasmic estrogen receptor and show therefore proliferative effects if stimulated with estradiol (Pratt and Pollak 1993). Furthermore, they may express insulin-like growth factor binding proteins (IGFBP) BP-2, BP-4, BP-5 if they are stimulated with anti- estrogens (Takahashi and Suzuki 1993). The proliferation of MCF-7 cells can be inhibited via tumor necrosis factor alpha (TNF alpha) treatment (Sugarman et al. 1985). Important to note for apoptosis related investigations is that MCF-7 cell do not express Caspase 3 (Janicke et al. 1998).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.5 MCF-7 breast cancer cells

Shown are MCF-7 cells (~90% confluent, passage 7) in a 10cm tissue culture dish. The picture was taken via phase contrast microscopy using a Zeiss Axio Vison microscope, 10x objective.

1.5 Proteasome activator 28 gamma (PA28γ)

PA28γ also known as REGγ or Ki antigen is encoded by the PSME3 gene on chromosome 17q21. The gene includes 19,376bp. PA28γ has four different isoforms. The predominat is isoform one with 254 amino acids (aa). PA28γ belongs to the family of PA28. Other members are PA28α and PA28β (NCBI 2016). However PA28γ shares only 25% in aa identity with PA28α and PA28β whereas the complementarity between PA28α and PA28β is about 50% (Li and Rechsteiner 2001).

One major protein degradation pathway is represented by the ubiquitin dependent proteasome system (UPS). The UPS is involved in the regulation of the cell cycle, death of cells, cell signaling transcription and immune responses (Zhang et al. 2004). The UPS also called 26S proteasome, needs ATP for the degradation of ubiquitin conjugated proteins. It is composed of the cylindrical 20S core and the proteasome activator 700 (PA700 or 19S regulator) (Voges et al. 1999). In the 19S regulatory particle are six different ATPase subunits localized that cleave ATP to unfold and deubiquitinate the protein targets before they are degraded (Pickart and Cohen 2004). Its counterpart is the PA28γ (11S regulatory particle) associated 20S proteasome. This is an ATP and ubiquitin independent system (Chen et al. 2007). The 20S core unit is cylindrical shaped and made up of 4 stacked heptametrical ring structures. Each of the inner two ring consist of seven β-subunits whereas the outer two rings are composed of seven α-subunits. These outer rings serve as docking points for the activator molecules and the β rings contain the protease active sites performing the proteolytic degradation (Smith et al. 2007). Three different proteolytic activities are discussed to be performed by the 20S core unit: chymotrypsin-like, trypsin-like and caspase-like activity, depending on the associated PA28 molecule (Wilk and Orlowski 1983).

For stabilization and binding to the 20S proteasome, PA28γ forms homo-heptametrical complexes (Kravtsova-Ivantsiv and Ciechanover 2012). REGγ activates the proteasome mediated cleavage after basic amino acid residues and enhances the trypsin-like activity of the 20S proteasome (Realini et al. 1997). However REGγ mutant K188D/E is able to activate all three activities but simultaneously destabilizing the PA28γ heptamer structure (Li et al. 2000). Ammonium sulfate precipitated REGγ showed the same characteristics as the K188D/E mutant. So this indicates an indirect proportionality between the stability of PA28γ heptamers and its ability to cross activate all proteasomal, proteolytic activities (Gao et al. 2004).

Structural analyses of PA28γ revealed four 33-45 residues long α-helices with a linker sequence between helix two and helix three. This linker is called “activation loop” due to the fact that a point mutation within this structure (N151Y) disables REGγ to activate the proteasome although it is bound to the 20S core unit. So it was showed that the linker sequence between helix two and three plays a critical role in proteasomal activation (Zhang et al. 1998). The C-terminus has been shown to be important for heptamer stabilization and proteasome binding (Li et al. 2000).

PA28γ is involved in cell growth regulation as suggested in studies dealing with REGγ knockout mice (Murata et al. 1999), additionally REGγ -/- mouse embryonic fibroblasts (MEFs) had a reduced number of S phase cells and a significant propagated tendency to become apoptotic (Barton et al. 2004).

In cancers PA28γ is often overexpressed and it is proposed to be a useful tumor marker in thyroid cancer (Okamura et al. 2003) and colorectal cancer (Roessler et al. 2006). REGγ is involved in the proteasomal degradation of oncogenic proteins as SRC-3, HCV core protein and PTTG1 but it also targets tumor suppressors like p21 or p53 (Sharpless and DePinho 2002, Moriishi et al. 2003, Li et al. 2006, Ying et al. 2006, Li et al. 2007). The 11S regulatory particle modulates E3 ubiquitin ligases e.g. murine double minute 2 (MDM2). MDM2 is so enabled to ubiquitinate p53 and promote its degradation by the 26S proteasome (Zhang and Zhang 2008).

Furthermore, PA28γ is described to be linked with DNA damage response (DDR) kinases Chk2 and ATM (Chen et al. 2005).

1.6 Preliminary work and aim of the study

1.6.1 Preliminary work and background of the study

In previous work the research group of Prof. Dr. Ralf Stohwasser investigated the role of the proteasome activator PA28γ as an anti-apoptotic regulator. They found that the sensitivity of cells towards apoptosis correlates with the cellular PA28γ levels and that PA28γ has strong effects to apoptotic hallmarks especially p53 phosphorylation and caspase activation. For this stable overexpressing PA28γ B8 mouse fibroblasts upon UV-C stimulation and also HT29 adenocarcinoma cells with silenced PSME3 gene upon butyrate treatment were analyzed (Moncsek et al. 2015).

After the investigation of the PA28γ pathway during cancer cell development by others it has been shown a differential regulatory effect by wild type p53 and p53 mutants signaling (Ali et al. 2013). Wild type p53 together with TGF-β is able to inhibit the REGγ-proteasome pathway and prevent so p53 from PA28γ/ MDM2 mediated ubiquitination and proteasomal degradation. This negative feedback loop is believed to exist between p53 and PA28γ if p53 fulfill its biological functions as triggering apoptosis (Ali et al. 2013). Several cancer models show a mutation in the tp53 gene which results in a gain of function (GOF) mutation. A GOF mutation of p53 results in an enhanced expression of REGγ and so also in an increased resistance to apoptosis or chemotherapy. Mutant p53 antagonizes with TGF-β/ Smad signaling and its function of suppressing PSME3 gene expression (Ali et al. 2013). The upregulated transcription of the PSME3 gene was also found by Wang and colleagues (Wang et al. 2015). They referred the ability of p53R248Q mutant to bind to the REGγ promotor region and recruit other still unknown transcription factors (Wang et al. 2015). It has also been shown that PA28γ knockout cells provided a lower proliferation rate, reduced migration and invasion. Interestingly, a p53R248Q transfection of REGγ knockout cells recovered the malignant characteristics (Wang et al. 2015).

1.6.2 Aim of the study

The aim of the study is divided into two parts.

1 At first the method of CRISPR-Cas9 should be established in the laboratory. For this purpose gRNAs targeting PSME3 exon1/exon4 as well as tp53 exon1_1/exon 1_2 should be designed and cloned into CRISPR-plasmids (pX330 and pX458). In the following MCF-7 cells should be transfected with the CRISPR plasmids to achieve a knockout of PA28γ and p53.

2 The second part aims at studying the transfected cells. Investigated should be the efficiency of the transfection, the quality of gRNAs, the knockout of the PSME3 and tp53 gene and the apoptotic behavior of modified MCF-7 cells.

2 Material and Methods

2.1 Materials

2.1.1 Laboratory equipment and instruments

Table 2.1 Laboratory equipment and instruments

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2.1.2 Plastic materials and consumables

Table 2.2 Plastic materials and consumables

Plastic material/consumables Supplier

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2.1.3 Chemicals, buffers and media for cell biology

Table 2.3 Chemicals, buffers and media for cell biology

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2.1.4 Chemicals, buffers and enzymes for molecular biology

Table 2.4 Chemicals and buffers for molecular biology

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Table 2.5 Enzymes for molecular biology

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2.1.5 Chemicals and materials for biochemistry

Table 2.6 Chemicals and materials for biochemistry

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2.1.6 Cell lines and bacteria

Table 2.7 Cell lines

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2.1.7 Antibodies

Table 2.9 Antibodies

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2.1.8 Kits

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