3. (4 pts) You have learned about a number of genes that play roles in Alzheimer

Question

3. (4 pts) You have learned about a number of genes that play roles in Alzheimer’s Disease (AD), such as uracil glycosylase, MED12, APP, PEN-2, and NEP. Pick one of these genes, and describe how could you use CRISPR-Cas9 to target this gene in mice in order to engineer a mouse that is MORE likely to develop AD (or something approximating it in mice). Specifically describe how you would use the CRISPR-Cas9 system and why the change you make in the target gene will increase the likelihood of AD.

Grading: 1 pt for describing what change you would make in the target gene; 1 pt for describing how you would design the CRISPR guide RNA (not the specific sequence, but the information you would need in order to decide on the specific sequence) and, if needed, the repair template DNA; 1 pt for indicating whether you would need non-homologous end joining (NHEJ) or homology dependent repair (HDR) to act on the DNA after Cas9 cleavage; 1 pt for explaining why the change you engineered would increase the likelihood of AD.

Explanation / Answer

Let us choose uracil glycosylase.

Uracil glycosylase gene is also known as Uracil DNA glycosylase (UDG). This gene codes for UDG protein. UDG protein prevents mutagenesis by eliminating uracil from DNA molecules. We know that uracil if present in DNA will bind to adenine; and will convert G-C base pair into A-T pair. It is important to note that this enzyme eliminates uracil from "AU" as well as "GU" base pairs.

Alzheimer’s Disease (AD) is an age related neurodegenerative disorder. In this disease Base excision repair enzymes are hampered. As a result, mispairing of uracil cannot be corrected. This results in mental retardation and memory loss in the persons having this disease.

CRISPR-Cas9 system is a gene editing tool. It removes or adds up DNA in gene; thereby promoting gene therapy (in a way).

In CRISPR-Cas9 system, two molecules are there.

Now, when gRNA is applied to the sequence, scaffold part of gRNA binds to DNA; and guides Cas9 to that place where cut has to be made. After the cut has been made by Cas 9, gRNA binds to that region; because its sequence is complementary to that region of DNA.

Cas9 makes a cut in both sides of the DNA. This cut is recognised by DNA repair machinery and correction is made in the next replication cycle.

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