Difference between revisions of "2016 Lab 4 - CRISPR/Cas9"

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3. Method (the design of your KO experimental procedure, referenced)
 
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 <ref><pubmed> 25733580 </pubmed></ref> Mice embryos identified with these single point mutations will undergo CRISPR methods to edit and correct this point mutation. This method of targeting a single point mutation was utilised by 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. <ref><pubmed>27010574</pubmed></ref>  
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Sickle cell disease involves a single point mutation in the seventh codon in the  β-globin gene <ref><pubmed> 25733580 </pubmed></ref> Mice embryos identified with these single point mutations will undergo CRISPR methods to edit and correct this point mutation. This method of targeting a single point mutation was utilised by 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. <ref><pubmed>27010574</pubmed></ref>  
  
 
4. Results (how the results could be interpreted/tested)
 
4. Results (how the results could be interpreted/tested)

Revision as of 13:23, 7 April 2016

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


Results:


Group 3

Group 4

Group 5

1. Hypothesis (the hypothesis you are testing)

That correcting the genetic mutation in mice embryos with sickle cell anemia can cure the disease in mice

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 <pubmed>8663884</pubmed>.. To use CRISPR to investigate whether genetically correcting the Sickle cell anemia can cure the disease when Rats develop in life.


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 [1] Mice embryos identified with these single point mutations will undergo CRISPR methods to edit and correct this point mutation. This method of targeting a single point mutation was utilised by 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. [2]

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


  1. <pubmed> 25733580 </pubmed>
  2. <pubmed>27010574</pubmed>

Group 6

Hypothesis (the hypothesis you are testing)

Aims

To determine if knocking out the Prion Protein gene affects susceptibility to multiple system atrophy (MSA)

  • Successfully knock out PrP gene
  • Infect all mouse 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)

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

Three groups Control (PrPC) protein and prion form of alpha synclein disease causing agent KO (PrPC) protein and prion form of alpha synclein disease causing agent



Group 7



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