Consider our yeast CRISPR-Cas9 experiment: yeast are transformed with the PML 104-9RNA1 plasmid + HDR1 template to edit the ADE2 gene. Enter your answers below. a. In yeast, what is the template from which the gRNA is transcribed?
b. The HDR fragment must enter the of the yeast cell to be used for homology directed repair.
c. The Cas9 gene is transcribed from the as template, the transcript is transported from the to the of the cell, and then the protein is translated.
d. After translation of Cas9, what happens? Explain in ONE sentence.

a. The guide RNA (gRNA) is transcribed from the PML 104-9RNA1 plasmid vector

b. It is correct. The homology-directed repair (HDR) fragment of interest enters into the cell and is then inserted at the site of the original double-strand breaks (DSBs) via the HR DSB repair pathway.

c. plasmid, nucleus, cytoplasm

d. Once Cas9 is transcribed, this nuclease forms a complex with the gRNA and cuts the target DNA, thereby inducing a DNA double-strand break (DSB) which is repaired either by non-homologous end joining (NHEJ) or homology-directed repair (HDR)

Explanation:

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (i.e., CRISPR-associated protein 9) is a natural prokaryotic defense mechanism against foreign viral invaders. In genome engineering, this system has been adapted to create a versatile RNA-programmable genome editing technique. Cas9 is an RNA-guided endonuclease capable of recognizing and cleaving a target DNA complementary to the guide RNA (gRNA). The gRNA is a specific RNA sequence formed by two small RNAs (crRNA and tracrRNA), which binds to the Cas nuclease enzyme and guides the gRNA-Cas9 complex to the target DNA sequence.

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