5. (15 pts) At the end of the semester we discussed DNA damage and repair system

Question

5. (15 pts) 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 describe the components of the gene editing system (answer here ii.) the in vivo or normal functions components of the gene editing system answer here) ii.) the steps in use of the system to introduce a double stranded DNA break in the target DNA (answer here) iv.) the steps in the cell's repair system(s) and how this leads to the edited DNA (answer here) v.) how are the components of the gene editing system inserted into cells? (answer here) vi.) 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. answer here)

Explanation / Answer

In case of CRISPR Cas 9

1. Components are - clustered regularly interspaced short palindromic or CRISPR , Cas 9 protein (CRISPR associated protein 9) and guide RNA or gRNA

2. CRISPR is short repetitive base sequences of bacteria which provides acquired immunity to fight against viruses. Cas 9 is DNA directed endonuclease enzyme. gRNA guides Cas 9 to bind at the required location.

3. In case of CRISPR Cas 9, the Cas 9 is the site specific double stranded endonuclease,which introduced to breaks the DNA double strand. gRNA guides the Cas 9 to reach the desirable site where Cas 9 break down the weak hydrogen bonds between two complementary DNA strands.

4. The steps of the DNA editing are as follows

First an endonuclease is introduced which break the desirable site of the target DNA.

Second, the DNA which will be inserted at the target location is introduced

Lastly the double stranded break repaired. It is done by either nonhomologous end joining or homologous recombination.

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