Difference between revisions of "2016 Lab 4 - CRISPR/Cas9"
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Latest revision as of 16:46, 26 April 2016
For Lab 4 class working in your Project Group, design an experiment employing CRISPR knockout technologies that would investigate a human disease.
The experiment should have:
1. Hypothesis (the hypothesis you are testing)
2. Aims (a series of specific aims of your experiment)
3. Method (the design of your KO experimental procedure, referenced)
4. Results (how the results could be interpreted/tested)
See also video JoVE Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Please paste your experiment under the appropriate group sub-heading below.
- 1 Group 1
- 2 Group 2
- 3 Group 3- Testing the effect of knockout of MLH1 gene as a model for Lynch syndrome carcinogenesis
- 4 Group 4
- 5 References
- 6 Group 5
- 7 Group 6
- 8 Group 7
- 9 2016 Course Content
Disease - β-thalassemia
The haematopoietic differentiation efficiency of HBB gene defective haematopoietic stem cells (HSCs) by CRISPR/Cas9 gene knockout system will be significant compared to HSCs that have not undergone treatment. The morphology of erythrocytes will be normal following injection of corrected HSCs into the bone marrow of diseased mice whilst the erythrocyte count and globin synthesis will be elevated following treatment. However, the treatment is unlikely to help the diseased mice reach the same conditions as healthy mice.
To determine the haematopoietic differentiation efficiency of gene - corrected beta - thalassemia haematopoietic stem cells by CRISPR/Cas9 gene knockout system in a mice model
To analyse the morphology and quantity of erythrocytes across the mice group
To determine and compare the levels of globin synthesis across different mice group
1. A blood sample is to be taken from healthy mice (Group 1) and mice known to be suffering from β-Thalassemia (Group 2) .
2. Both groups will have their blood samples taken and the erythrocyte count (on blood film) and morphology will be recorded.
3. β- globin synthesis will be measured using western blotting and recorded
4. Haematopoietic stem cells (HSCs) will be extracted from the bone marrow of mice in group 2.
5. The CRISPs/Cas 9 system purchased from ORIGENE Cat# GE100010 will then be employed to knock out the defective HBB gene.
6. Replacement vectors will then be used to integrate functional HBB genes into the HSCs and left to be incubated in fresh medium.
7. The genetically corrected HSCs are then injected back into the bone marrow of mice in Group 2 and be allowed to recover.
8. After 4 weeks, blood samples will be taken from Groups 1 and 2 and the erythrocyte count, morphology and expression of β- globlin chain will be recorded and compared.
Erythrocyte count from before and after treatment will be compared between the treated mice and positive control mice (healthy). Theoretically, after CRISPR deletion of the mutated gene and healthy gene transfection, erythrocyte concentration should increase within a few weeks, with their morphology returning to normal. We should find that treated mice (Group 2) will have a higher red blood cell count than before it was treated and the erythrocytes should display normal morphology compared to when there was microcytic anaemia. The erythrocyte count is expected to be less than the positive control mice before and after the treatment, since the β-Thalassemia is unlikely to be reversed in Group 2.
Using western blotting, we measure the expression levels of β- globlin in both groups of mice before and after the injection of haematopoietic stem cells. We then compare this expression. It is expected that the expression of β- globin in the treated thalassemic mice will be lower compared to the healthy group. However, the levels of β- globin synthesis would be much higher in the diseased mice following treatment with HBB gene corrected HSC mice.
Ciavatta, D. J. et al. "Mouse Model Of Human Beta Zero Thalassemia: Targeted Deletion Of The Mouse Beta Maj- And Beta Min-Globin Genes In Embryonic Stem Cells.". Proceedings of the National Academy of Sciences 92.20 (1995): 9259-9263. Web.
Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome research. 2014 Sep 1;24(9):1526-33.
Song B, Fan Y, He W, Zhu D, Niu X, Wang D, Ou Z, Luo M, Sun X. Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem cells and development. 2014 Dec 17;24(9):1053-65.
In general, hypotheses and Aims aligned. Methods also address the Aims. Results section - quite good understanding of what sort of data will be produced and implications.
Knocking out the SEC23B gene in mice will result in the development of an anaemia similar to Congenital Dyserythropoietic Anaemia Type 2 in humans
Utilise CRISPR technologies and methods to knockout the SEC23B gene in mice
Analyse the nuclei of the erythroblasts
Analyse the morphology of erythroblasts and mature red blood cells
Analyse the haemoglobin levels in the blood spectroscopically
Compare all results with the control subjects
16 newborn mice, equal numbers of male and female, and half had their SEC23B gene knocked out using the CRISPR method while the other half remained genetically normal.
Cas9, in a electroporation compatible buffer, was sourced from Supplier A
All controls were subject to electroporation followed by injection of saline solution. They were also analysed using the same methods for the knockout mice
Methods and Techniques
CRISPR: Place the Cas9 into the buffer and suspend into a cuvette. Shave an area of fur close to the target bone marrow, anaesthetise that area and inject the cas9-buffer into the bone marrow. Following this, apply the appropriate voltage to electroporate the cells
Leave the mice for 2 days then extract 5mL of blood from the bone marrow of each mouse and centrifuge to separate the red cells. Examine the morphology of the erythrocytes and erythroblasts using light microscopy, looking for abnormalities such as multiple nuclei and irregular cell shape.
Extract another 5mL of blood, centrifuge and place the red cells into an automated haemoglobin spectrometer to analyse the haemoglobin levels. Compare this result with the normal mice.
The use of Crispr cas-9 on erythrocytes present in the bone marrow would result in an increase of bi- and multi-nucleated erythrocytes with a high amount of DNA present within the nuclei, therefore suggesting a possible defect in erythrocytic cytokinesis. It can be suggested that a sufficient level of SEC23B is required for efficient cellular replication and decreases in abnormalities. Morphological structures of erythrocytes can be observed through light microscopy. Possible cellular abnormalities observed would include abnormal cell shape and size. Erythrocytes present in SEC23B deficient mice would lose their biconcave shape and also have reduced surface area.
With the increased number of nuclei present within the erythroblast, this would leave less room available for haemoglobin, therefore also having a decreased amount of haemoglobin present within the cell leading to decreased oxygen carrying capacity. Levels of haemoglobin is measured using spectroscopy. The levels of haemoglobin were compared to control mice (with SEC23B gene present) and experimental mice (with SEC23B deficient). SEC23B deficient mice would present lower levels of haemoglobin compared to normal mice with the SEC23B gene expressed normally.
Hypothesis - question to be address clear. Aims - Overall Ok, but last aim is not an Aim as one would be comparing to controls in all the aims. Materials/Controls/Methods - lacking detail, no mention that mice will be made and hard to understand logic of experimental plan. Results - Good description of expected results. Too few references.
Group 3- Testing the effect of knockout of MLH1 gene as a model for Lynch syndrome carcinogenesis
Inactivation of MLH1 will prevent the MLH1/PMS2 protein complex from carrying out mismatch repair, a characteristic of Lynch syndrome, leading to an increased risk of cancer. The other major mismatch repair genes (e.g. MSH2/MSH6) will be up regulated.
To investigate knockout of the MLH1 gene and its significance in Lynch syndrome colon cancer carcinogenesis. To test whether other mismatch repair genes are upregulated in response to a faulty MLH1/PMS2 protein complex.
The plasmid expressing hCas9 was attained from ADDGENE repository gRNA was designed to target the MLH1 gene in a mouse embryo. 10 embryos were collected from mature female mice using the methods described by Kaneko and Mashimo The gRNAs and Cas9 were cloned into plasmids and then introduced into 5 of the embryo cells by transfection using the “Ingenio Electroporation solution and kit” , acting as the experimental. The other 5 embryos were not transfected. The GFP was introduced to select for cells that had taken up the plasmids through the “pGLO bacterial transformation kit” 
The embryos were cultured in the reagents described on “Thermofisher scientific” 
Immunohistochemistry was used to measure the abundance of MLH1/PMS2 and MSH2/MSH6 protein complexes in the colon epithelial cells. A microarray analysis was carried out to measure the expression of various genes involved in mismatch repair. This was done at every week for a period of 6 months.
Immunohistochemistry staining specific to MLH1/MSH2 and MSH2/MSH6 indicates the presence or absence of these proteins and their relative abundance in the colonic epithelial cells. Positive staining indicates that the complexes are present; negative staining indicates that they are scarce. If the experiment is consistent with the hypothesis, staining for MLH1 will be negative, PMS2 staining will be positive but no change between control and experimental, staining for MSH2/MSH6 will be positive and in the experimental compared to control. Microarray analysis indicates whether other genes involved in carcinogenesis, including other mismatch repair genes, are upregulated or downregulated in the colon epithelial cells. A graph of experimental vs. control fluorescence will increase in gradient, indicating upregulation of MMR genes.
Hypothesis - Good, very clear and precise. Aims - Also v. good, aligns with hypothesis. Methods - quite hard to understand the logic of the experimental plan. Would mice be analysed or just the embryos, etc. Results - quite good idea of what results would come from the analyses. References - rely too much on webpages. Need peer reviewed journal articles.
CRISPR induction of the KIR positive haplotype 1 (2dl5) gene causes a 16 fold decrease in risk for rheumatoid arthritis comparative to the KIR negative haplotype 6 gene.
To study the effect of positive KIR 2dl5 (haplotype 1) gene expression induction in healthy humans adults, over the age of 30 using CRISPR and its association with RA.
- screen adults over 30 for the negative KIR haplotype
- induce double positive KIR gene expression in half of the sample size
- observe the long term impact of this gene alteration, specifically on RA
Screen 400 patients using PCR/SSP (Polymerase chain reaction/specific-sequence primers)  PCR with specific sequence primers will identify the presence or absence of the positive KIR 2dl5 gene. Use CRISPR to isolate KIR haplotype 1, and mAB to cultivate this sequence in mice. 
Referring to Table 5 in a study by Nazari.M et al. KIR Haplotype in healthy individuals and patients with RA, we predict that induction of a double positive KIR haplotype will lead to a decrease in RA presentation in healthy individuals over time. It will be highly unexpected that there will be no correlation between a double positive KIR haplotype and RA presentation as there is statistical significance of <0.0001.
Hypothesis - outcome hypothesized too specific - 16-fold decrease; significant decrease better. Aim - mostly methods not Aims. Methods - very sparse with details and not clear how this will address hypotheses. Results - also not particularly clear of what the expected results will be.
Sickle cell disease is a debilitating genetic disease that's only cure is the heavily invasive bone marrow transplant . Developing a prevention strategy such as in vivo genetic correction would be the key to curing this disease. The aim of this experiment is to use CRISPR to investigate whether genetically correcting the Sickle cell anemia point mutation can cure the disease in vivo.
That mice that have the human sickle cell anemia gene can undergo CRISPR to correct the mutation in vivo.
Sickle cell disease involves a single point mutation in the seventh codon in the β-globin gene in humans.  As in other studies , human induced sickle mice will be analysed. Those identified with the pathogenic single point mutation will undergo CRISPR methodology to edit and correct this point mutation. The method that will be used for the genetic recombination of the embryos will mirror the Hampton et al. in their study CRISPR-Cas gene editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia, which edited the point mutation in fLhC.  As a control, a group of wild type mice will also undergo analysis using the same CRISPR techniques.
The results for this experiment can be tested and interpreted using PCR amplification. Samples of DNA will be taken from the bone marrow of the mice to be amplified. PCR amplification is able to detect and validate the gene edit that we performed during the experiment and can also be used to analyse the effects of the edit. 
- <pubmed> 25733580 </pubmed>
Hypothesis and Aims: very nicely stated. Clear, to the point and precise. Methods: didn't actually state that mice will be made. Would have been good to have more details of tests that will be performed on the mice. Results - again too little detail and nothing about what is expected about the mouse phenotypes. References - a number of very relevant journal articles - v. good.
It may be possible that cellular prion protein (PrPC), associated with Creutzfeldt–Jakob disease, is related to other neurological disorders in which prions are implicated. Our hypothesis is that PrPC and its activity within the cell is instrumental in the proposed replicative characteristics, and thus pathogenicity, of alpha-synuclein protein. CRISPR will successfully knock out the gene for PrPC, and it is hypothesised that the resulting mice will display less pathogenic symptoms than the infected mice with the gene for PrPC knocked out.
To determine if knocking out the (PrPC) gene affects susceptibility to multiple system atrophy (MSA)
- Successfully knock out (PrPC) gene
- Infect all non-control mice with prion form of Alpha-synuclein
- Quantify the amount of misfolded alpha-synuclein protein in knock out and healthy mice
CRISPr/Cas 9 kit was obtained through Australian bioresources
Mice neuroblastoma cells were obtained through ATCC
"CRISPR design tool" was used to determine appropriate target sites within the (PrPC)
Generation of gRNA Expression Vectors, Cell Culture and Transfection, Generation of stable knockdown cell clones, Genetic analysis, Western blot analyses were done as in CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome 
As in *Neuropathology in mice expressing mouse alpha-synuclein*, for all three groups, control, PrPc and koPrPC we will use an anti alpha synuclein antibody from (Syn-1, 1∶500; S63320, Transduction Laboratories and 4D6, 1∶800; Abcam) which we will visualize with avidin-biotin peroxidase method (Elite standard kit SK6100, Vector) and DAB substrate (1718096 Boehringer). And use a confocal microscope (Leica TCN NT) to infer fluorescence and compare the fluorescence of all three group.
We have three different groups, which are Control, PrPC with prion form of alpha synuclein disease causing agent, and KO PrPC with prion form of alpha synclein disease causing agent.
The results that we can expect are that the amount of alpha-synuclein protein found in mice with active PrPC protein will be greater than the mice with KO PrPC protein. However, we expect that both of the alpha-synuclein protein groups will report more amount of alpha-synuclein than the control group. This result could show that PrPC is involved in the function of the pathogenesis of prion diseases.
- <pubmed> 25490046</pubmed>
Hypothesis and Aims: The question to be tested clearly outlined. Also indication of the clinical problem to put into context. Methods: Not too bad, but a bit unclear (lacking in detail) of what will be done with the mice. Results - again quite good. Seem to understand what results can be expected. References - v. good list of relevant journal papers.
Mutation of the methionine codon at position 129 of the allele of the PRNP gene (which codes for PrP protein) reults in a rare prion disease; Fatal Familial Insomnia. The aim of our experiment is to determine the role of the PrP protein in normal conditions in the brain by use of the CRISPR KO method in an animal model to show the effect of absence of the protein in the hippocampus and its effect on spatial memory, tested through maze trials. 
It is expect that the absence of PrP will show diminished spatial memory on the second maze trial compared to mice with PrP.
CRISPR/Cas9 method  was used to knock out the PRNP gene at the short (p) arm of chromosome 20 at position p13 in germ cells of mice and a non-coding sequence was inserted. The ES cells that accepted the insertion were selected through treatment with neomycin then ganciclovir and inserted into the blastocysts of pseudopregnant mice. Untreated mice with the normal gene were used as a control. Both treated and untreated mice were placed in a maze with a reward at the end and were timed on their completion of the maze. Two days were allowed to pass before the mice were again placed in the same maze and times recorded.
- Expected It is expected that the treated mice without the PrP protein will show longer times upon completion of the second trial of the maze compared to the control mice. 
- Unexpected There will be little to no difference in completion times between treated and untreated mice upon the second completion of the maze.
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Hypothesis and Aims: Clearly outlined. Methods: Concise but well described. The logic of the experiments well outlined. Results: a bit limited, but what was mentioned is clear and logical. References: good listed of relevant journal articles.
2016 Course Content
Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | 2016 Revision
Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Cell Knockout Methods | Cytoskeleton Exercise | Immunochemistry | Project Work | Confocal Microscopy | Tissue Culture | Stem Cells Lab | Microarray Visit
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G