Question

At the end of the semester we discussed DNA damage and repair systems. Then we read some papers on gene editing tools because these tools depend on DNA repair mechanisms in the cell to complete the editing process. For one of the three major editing tools, ZNFs (Zinc finger nucleases), TALENS (transcription activator-like effector nucleases) or CRISPR Cas9 answer the following:
     i. What are the components of the gene editing system? (2 pts)
           (type answer here)

    ii. What are the in vivo or normal functions components of the gene editing system? (2 pts)
           (type answer here)

    iii. What are the steps in use of the gene editing system to introduce a double stranded DNA break in the target DNA? If you include figures to help you answer this question be sure to insert in this section with your typed answer. (3 pts)
           (type answer and insert figure here)

     iv. What are the steps in the cell’s repair system(s) and how does this participate with these gene editing systems, leading to the edited DNA? If you include a figure to help you answer this question be sure to insert in this section with your typed answer. (3 pts)
           (type answer and insert figure here)

     v. How are the components of the gene editing system introduced to or inserted into cells? (2 pts)
           (type answer here)

     vi. Give a specific example of use of this gene editing system in attempt to correct the error(s) of an inborn error of metabolism. Describe how this was used to edit DNA to alleviate a defect and identify whether this was an in vivo, in vitro or ex vivo application and if successful (2 pts).   
       (type answer here)

References used to answer the parts of this question

Answer 1

Answer i

Zin Fingers has two domains of unique hexamers that uses to bind with the targeting DNA and one nuclease enzyme Fok 1.

Answer ii

The domains bind with DNA and form a protein complex with the DNA. The protein acts on the complex and DNA gets a double strand break.

Answer iii

First, the recognizable hexamers of the Zinc fingers complex recognize the DNA strand and bind. The protein enzyme linked with the hexamer through a linker binds on DNA and make dimer. Each zinc finger has monomer of the Fok 1protien, which must dimerize to make the cleavage. That cleavage is double DNA break. The protein is made of 30 amino acids.

Answer iv

• Prepare Zinc fingers, desired DNA with the vector like P element.
• Isolate cell host and culture them.
• Insertion of Zinc fingers can obtain through any of the heat shock, embryo injection, zygote injection, or protoplasts injection and several other methods. This helps insertion of vector to the cell.
• After functioning of the enzyme, the desired DNA will insert into the DNA of the host.
• Screen the desired cells and grow them on culture medium.

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