On this page, we will clarify what our project goal is, and show our material selection, component design and experimental design, and characterization methods designed to verify the results in order to successfully achieve this goal.
It should be noted that this page focuses on the experimental design, and the consideration of the rigor of the characterization method and the reliability analysis of the results will be discussed in Measurement.
Our project revolves around how to obtain chromosome-free eukaryotic cells (CREATE), and the following experimental purposes are determined step by step:
(1) Construction of CREATE
(2) Characterization of the formation of CREATE
(3) Exploration of the features of CREATE
With these goals in mind, we will introduce our experimental design in detail in the following sections.
Construction of CREATE
We use CRISPR/Cas9 system to achieve multiple double-stranded DNA breaks at multiple copy sites in yeast genome, and then spontaneously complete chromosome degradation under the action of endogenous nuclease.
Selection of engineering strains
Our project aims to construct and verify chromosome-free eukaryotic cells and explore their properties and applications.
Eukaryotic cells are chosen because of their cellular mechanisms, such as organelles, which make them more perfect and have greater application potential. In eukaryotes, we finally chose Saccharomyces cerevisiae as our engineering strain. As model organisms and commonly used engineering strains in eukaryotic microorganisms, we know more about their cellular mechanisms and can borrow more toolboxes.
Choice of chromosome degradation methods
We looked up some literature on chromosome destruction and got some methods to degrade genomes.
There are methods not selected and the reasons:
×Use endonuclease to cut the corresponding cleavage site
Within the genome, there may be many recognition sites of endonuclease. Therefore, endonuclease can be used to make multiple DNA double strand breaks (DSBs) in genome, and the rapid degradation of genome can be realized with the help of endogenous Dnase. However, this method is suitable for Escherichia coli with small genome, but not for eukaryotes with complex genome structure. At the same time, yeast has strong homologous recombination repair ability, which makes inefficient cutting methods unable to complete chromosome degradation. Selecting and expressing hetero nuclease is not an easy task, and this method is not the most suitable method for our project.
×Cut specific regions of chromosomes
Some literatures show that a gap can be created near the centromere of chromosome, which will easily lead to the degradation of the whole chromosome. However, there are 16 chromosomes in yeast. If we choose this method, we need to construct different cutting elements according to the differences of each chromosome, which will lead to complicated construction.
Final selection method:
√Use CRISPR system to cut:
Because yeast has strong homologous recombination repair ability, we need a cleavage system that can produce as many double strand breaks as possible in the whole genome in a short time. On the other hand, its cleavage efficiency should be efficient enough. After the above analysis, CRISPR technology has entered our field of vision. As a widely used system, its high cutting efficiency and simplicity of use have been fully proved.
Select cutting sites:
By consulting the literature, we found a multi-copy locus with high-fidelity in yeast genome-Delta locus, which is highly homologous and uniformly distributed in yeast genome. This means that only one sgRNA can be used to target these sites at the same time, so that sgRNA and Cas9 complex can quickly and effectively produce multiple DSBs on the whole genome. Moreover, it can be seen from the literature that if the chromosome cannot be repaired in time, the broken fragment will be rapidly degraded by endogenous Dnase.
Figure 1 Delta-specific guide RNA
Design cutting system components
Cutting system 1.0--(1) Delta plasmid:
We initially constructed Delta plasmid. The sgRNA of Delta plasmid can target Delta site, which is the origin of the name of this element. Delta locus is a homologous, multi-copy homologous, multi-copy homologous sequence in yeast genome. Under normal circumstances, Cas9 protein on Delta plasmid is controlled by inducible promoter pGal and will not be expressed, while gRNA is constantly expressed. After induction, the complex formed by the two can target all Delta sites on yeast chromosome, thus creating multiple DNA double strand breaks (DSBs), which makes yeast genome unable to repair chromosome abnormalities smoothly, and will eventually cause DNA fragment cleavage (endogenous nuclease will degrade DNA) to complete chromosome degradation.
Figure 2 Delta plasmid
Figure 3 Delta plasmid(schematic)
★Cutting system 2.0-(2) 7flip plasmid and Cre recombinase plasmid:
We designed a new cleavage system to improve Delta plasmid, which uses dual regulation mechanism to regulate cas9 protein expression more strictly.
This element consists of two loxP sites with different directions and inverted pGal promoter.
Figure 4 Schematic diagram of 7flip
Figure 5 Schematic diagram of 7flip working system
The transcription direction of pGal promoter in normal state is reversed, and there is a loxP site on each side of pGal promoter with the direction opposite. When we use estrogen to induce the expression of Cre plasmid, the expressed Cre recombinase can reverse the direction of pGal promoter. After galactose was used for induction, cas9 protein could be turned on for expression.
Characterization of the formation of CREATE
We designed a variety of methods from different angles to confirm the formation of CREATE.
Nucleic acid dye staining
We use nucleic acid dyes to intuitively reflect chromosome degradation. Hoechest dye is a membrane permeable nucleic acid dye, which can bind to DNA and emit strong fluorescence signal. We stained the cells with this dye and observed them under fluorescence microscope to directly verify whether the DNA of the cells disappeared.
GFP& fGFP fluorescence signal attenuation
We use the change of nfGFP fluorescence signal to indirectly reflect chromosome degradation.
nfGFP is a kind of GFP designed by us, which can be degraded rapidly and has a short half-life. We compared its degradation rate with that of normal GFP.
We integrated nfGFP element into yeast chromosome by homologous recombination.
Figure 6 fGFP fluorescence signal attenuation
By comparing the changes of bacterial growth trend, the degradation of genome was proved.
OD600 can reflect the number of bacteria to a certain extent. By measuring the changes of OD600 values of CREATE and normal cells, we obtained the growth curve trend of both cells. Unlike the normal growth control group, CREATE cannot grow and reproduce because its chromosome is cut. We assume that its growth curve should be maintained in a very low range, and it is difficult to rise. Through this distinction, we can explain the great difference in reproductive ability between the two groups of cells under the same treatment conditions, which indirectly proves the formation of chromosome-free cell.
Flow cytometry and sorting
We use flow cytometry to analyze and sort CREATE.
Flow cytometry is a device that can analyze and sort cells.
It can measure and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort designated cells from cell subsets according to preselected parameter range. We set up a variety of CREATE characteristic parameter signals, and through these signals comprehensive analysis and screening out CREATE. More detailed information can be found in measurement, please click here.
In order to verify the reliability of flow analysis and separation technology, we also designed the following two experiments.
(1) 96-well plate cell culture experiment:
Chromosome-free eukaryotic cells (CREATE) obtained by flow cytometry and normal cells were cultured in 96-well plates. From 1 cell per well to 1000 cells per well, the number of cells in the well is set in a gradient.
CREATE can't grow and reproduce after losing chromosomes. If the machine sorts well, the cells in each well in the 96-well plate will not grow (or only a few wells will become turbid), while the normal cells will grow normally.
Figure 7 Schematic diagram of experiment
(2) Cell growth experiment in solid medium:
The isolated CREATE cells and those of the control group were separated into single cells by use of cell dissector and cultured in the same solid culture medium.
If they are indeed eukaryotic cells without chromosomes, they cannot grow and reproduce, while normal cells in the control group can.
Figure 8 Schematic diagram of experiment
We use the growth status of bacteria on solid medium to prove genome degradation.
Coating plate counting experiment:
We added an inducer to the shake flask of the experimental group to induce Cas protein expression to cut chromosomes to form the chromosomal-free eukaryotic cell CREATE, and no inducer was added to the shake flask of the control group.Cultivate together under the same culture conditions. Take samples from the experimental group and the control group every 12 hours to measure the OD600, and dilute with double distilled water at the same multiple. After dilution, apply the same amount of liquid to the solid medium. The chromosomal-free eukaryotic cell CREATE cannot grow and reproduce, but the control group treated with the same conditions should grow normally.With the prolongation of the induction time, the number of colonies grown on the solid medium between the experimental group and the control group differed significantly.Through this distinction, we can indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.
Point dilution plate:
When doing the CFU experiment,we use the diluted bacterial solution for the Point dilution plate experiment.The experimental group and the control group take the same amount of bacterial solution (2ul), and spot the bacterial solution on the same piece of solid medium. Note that the bacterial solution diluted in the same multiple should be spot-painted on the same line.After the experimental group induces Cas protein expression and acts on the genome, it will have a certain impact on the cell growth state, but this will not happen in the control group. Therefore, we can observe the different growth status between experimental group and the control group based on the results of the point dilution plate.This indicates that the part is in working condition.
Exploration of the features of CREATE
In order to investigate whether the shape of CREATE cells will change after chromosome cutting, we used microscope to observe the cell morphology of CREATE cells sorted and purified by flow cytometry. You can click this link to view the results (link)
Metabolic activity and life span
In order to explore whether the enzymes in chromosome-free eukaryotic cells (CREATE) are still active, we designed the following experiments:
We use the generation and duration time of fluorescence signal to reflect the presence or absence of cell metabolic activity and the retention time of activity.
We designed a mcherry plasmid induced by anhydrous tetracycline (ATc) and characterized it in normal cells to verify the normal and stable expression intensity of its fluorescence signal.
Figure 9 Mcherry pattern diagram
Subsequently, we transformed mcherry plasmid into yeast cells together with cutting system, and prepared them into chromosome-free cells.
After that, we induced the plasmid to express mcherry.
If the enzyme is active in chromosome-free cells, then gradually brightened mcherry fluorescence will be observed after induction, and the time for CREATE to maintain metabolic activity can be roughly obtained by the time when mcherry fluorescence disappears.
Figure 10 Schematic diagram of experiment
In the experiment, we found that many conditions will affect the formation efficiency of chromosomes. In the future, we will continue to explore the effects of different strains, different induction time and other treatment conditions on chromosome-free formation rate. At the same time, we will strive to further improve the speed and efficiency of chromosome degradation, and further prolong the time for chromosome-free to maintain metabolic activity.
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 Shi Shuobo,, Liang Youyun, Ang Ee Lui & Zhao Huimin. (2019). Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.. Methods in molecular biology (Clifton, N.J.) (), doi: 10.1007/978-1-4939-9142-6_6.
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