You are working in a lab and have some free time to do extra research. You decid

Question

You are working in a lab and have some free time to do extra research. You decide to launch your own mini project studying Huntington's disease. In unaffected individuals, the Huntington gene has 26 or fewer CAG repeats (each CAG repeat codes for a glutamine). Individuals with Huntingtin's disease have 36+ CAG repeats. You want to determine the role of the glutamine repeats in a wildtype cell. To do so, you decide to delete all CAG repeats from the Huntington disease in a mouse using CRISPR. 2. You need to first check and see if you successfully removed all the CAG repeats with CRISPR. In 442, you learned 4 DNA-focused methods (PCR, ASO, PCR primer initiation, and restriction site analysis) that you could use. After obtaining a sample from the mouse and isolating genomic DNA, explain the steps required for 2 of the techniques (include the expected result if your CRISPR was successful and removed the CAG repeats in both allele of Huntington - label it "expected result"). The first result is provided for you:* i.Option 1 - PCR Amplify the region of interest using PCR with flanking primers (before & after the tandem region) Run the PCR product on a gel. Compare the length to the same reaction done with the wt gene (should be 78 bp shorter)» Expected result: if the CAG repeats were removed, the CRISPR PCR product should be 78 bp shorter than the wt a. b. c.

Explanation / Answer

In Huntington disease, an increase in the size of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. The dysfunction and eventual death of neurons in certain areas of the brain underlie the signs and symptoms of Huntington disease.

To remove the CAG repeats in the CRISPR knock out models, the following steps will be required-

1.Selection of target site, assays, and oligonucleotides, as well as predicted off-target sites. Preparation of sgRNA (Subgenomic mRNA's are essentially smaller sections of the original transcribed template strand).

2.Injection of sgRNA (Subgenomic mRNA's are essentially smaller sections of the original transcribed template strand).and Cas9 mRNA into fertilized embryos, to generate FO mutated candidate animals. Breeding and genotyping to identify FO animals with the desired mutation.

3.The new founders are bred with wildtype mice of matching strain background, and their offspring genotyped to identify F1 mice bearing the knockout or knockin allele.

We should study the m-RNA expression of the knockout mice and perform RNA isolation (from mice tissue or blood) and RT-PCR analysis and gel electrophoresis of the DNA. So, if the CAG repeats were removed, the product should be shorter, like that of normal.

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