2016 Lab 4 - CRISPR/Cas9

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DNA targeting platforms for genome editing

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)


Search: NCBI databases - CRISPR | PubMed CRISPR | PubMed Centrap CRISPR

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.

Group 1

Group 2

Hypothesis: Knocking out the SEC23B gene in mice will result in the development of an anaemia similar to Congenital Dyserythropoietic Anaemia Type 2 in humans

Aims:

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, and compare with the normal phenotype

Analyse the haemoglobin levels in the blood spectroscopically


Methods:

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.

Materials Cas9, in a electroporation compatible buffer, was sourced from Supplier A

Controls All controls were subject to electroporation followed by injection of saline solution. They were also analysed using the same methods for the knockout mice

Techniques Place the Cas9 into the buffer and suspend into a cuvette. Following this, apply the appropriate voltage to electroporate

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.

Results:


Group 3

Group 4

Hypothesis

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.

Aim

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

Method

Screen 400 patients using PCR/SSP (Polymerase chain reaction/specific-sequence primers) [1] 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. [2]

Results

References

  1. <pubmed>26658904</pubmed>
  2. <pubmed>17371997</pubmed>

Group 5

2. Aims (a series of specific aims of your experiment)

Sickle cell disease is a debilitating genetic disease that's only cure is the heavily invasive bone marrow transplant [1]. 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.

1. Hypothesis (the hypothesis you are testing)

That mice that have the human sickle cell anemia gene can undergo CRISPR to correct the mutation in vivo.



3. Method (the design of your KO experimental procedure, referenced)

Sickle cell disease involves a single point mutation in the seventh codon in the β-globin gene in humans. [2] 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. [3]

<pubmed>18502677</pubmed>

4. Results (how the results could be interpreted/tested)

The results for this experiment can be tested and interpreted using PCR amplification. PCR amplification would be 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. [4]

  1. <pubmed>8663884</pubmed>
  2. <pubmed> 25733580 </pubmed>
  3. <pubmed>27010574</pubmed>
  4. <pubmed>24901507</pubmed>

Group 6

Hypothesis (the hypothesis you are testing)

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,[1] and it is hypothesised that the resulting mice will display less pathogenic symptoms than the infected mice with the gene for PrPC knocked out.

Aims

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

Method (the design of your KO experimental procedure, referenced)

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 prnp

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 [2]

Results (how the results/outcomes could be interpreted/tested)

We have three different group, which are Control, (PrPC) and prion form of alpha synclein disease causing agent KO(PrPC) and prion form of alpha synclein disease causing agent. We believe that form of the alpha-synuclein protein causes multiple system atrophy, and also research supports that (PrPC) function can play role in pathogenesis of prion diseases. The results that we can expect are the amount of alpha-synuclein protein can be found in mice with present of (PrPC)protein, while the mice with KO(PrPC)protein could find lesser amount of alpha-synuclein. However, both groups report more amount of alpha-synuclein than the control group. This result could show that (PrPC) is function in pathogenesis of prion diseases.

References

  1. <pubmed>20056882</pubmed>
  2. <pubmed>4260877</pubmed>

Group 7

Aims

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.[1] 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.[2] [3]

Hypothesis

It is expect that the absence of PrP will show diminished spatial memory on the second maze trial compared to mice with PrP.

Method

CRISPR/Cas9 method [4] 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.

Results

  • 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. [2]
  • Unexpected There will be little to no difference in completion times between treated and untreated mice upon the second completion of the maze.
  1. <pubmed>24275071</pubmed>
  2. 2.0 2.1 <pubmed>15837581</pubmed>
  3. <pubmed>20133875</pubmed>
  4. <pubmed>26857612</pubmed>

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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


2016 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G