Question

In: Biology

Most mammalian cell types do not undergo homologous recombination (HR), and even mouse ES cells do...

Most mammalian cell types do not undergo homologous recombination (HR), and even mouse ES cells do so at low frequency - about 1/200000 cells. However, if you first introduce a crRNA designed to target a mouse genomic sequence next to a PAM site, plus a source of Cas9 protein and a plasmid with homology arms, most mouse cell types will undergo HR with very high frequency -as high as 1/5 or more of the treated cells. What does this tell you about the mechanism of HR in mammalian cells?

A) The critical first step in HR is introduction of a double strand break at the target gene site.
B) Mouse ES cells have DNA repair systems that are defective compared to other cell types.
C) Cas9 protein directly activates expression of HR system genes in mammalian cells.
D) Mouse ES cells contain a larger number of PAM sites than most cell types.
E) Mouse ES cells already express higher levels of Cas9 than most cell types.

Solutions

Expert Solution

Homologous recombination or HR is a rare event. This has to be so to ensure that the genetic code is reliable and does not undergo frequent fluctuations.

However, as explained when Cas9 protein and crRNA for a PAM site are added alongside a plasmid with homology arms, the frequency of recombination increases. The reason for this lies in the function of Cas9.

crRNA is the complementary RNA for PAM site. And Cas9 is a Caspase 9 enzyme that acts as a nuclease. It binds to the crRNA and is thus guided to the PAM site. The Cas9 then chops off the DNA at the site resulting in a double-stranded DNA break.

This triggers the DNA-damage system. The DNA-damage system recognizes the presence of a homologous sequence and uses the Homologous recombination to repair the damaged site.

Thus, this experiment tells that the critical step in HR is the introduction of a double-stranded break. These breaks are not usually present in the cell.

Hence, the correct answer is A.


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