Explain clearly what components of the CRISPR-Cas9 system (and any other molecul

Question

Explain clearly what components of the CRISPR-Cas9 system (and any other molecules) would need to be delivered to hepatocytes to result in expression of the wild-type Fah protein. Include in your explanation the roles of each component in the correction of the Fah mutation. Even if you were to assume very efficient (100%) delivery of the CRISPR-Cas9 components to hepatocytes, explain why expression of the wild-type Fah protein would be expected to be at such a low level (1/250 cells).

If you do not know, DO NOT ANSWER. Please explain clearly and bluntly. :) I need these points.

Explanation / Answer

CRISPR -Cas9 mechanism is present in prokaryotes. When a phage infects a bacteria, say Streptococcus, the bacteria produces a nuclease Cas9, which helps it to cut the foreign nucleic acid molecule entering it. In very simple words, we can say that, CRISPR is a part of bacterial immune system; the cellular machinery which a bacteria uses to defend itself from virus.

Now, this technology has been used to correct Fah mutation in mouse hepatocytes. How??

Fah mutation causes defect in FAH gene; this results in Type I tyrosenemia; a rare disease in which the patient is unable to break down the amino acid tyrosine. Hence, tyrosine accumulates in the liver, finally leading to death.

In order to correct the Fah mutation, the following components are delivered into hepatocytes:

Cas9 unwinds the hepatocyte (foreign) DNA and the finds out complementarity to the 20 basepair spacer region of the guide RNA. If the DNA substrate is found to becomplementary to the guide RNA, Cas9 cleaves the invading DNA.

This approach was enough to cure the disease. In 1 out of every 250 cells, the corrected gene was introduced. This many cells account to most of the liver cells. Now, when healthy cells, will proliferate, the diseased cells will further be replaced. In about a month, 1/3 of liver cells were corrected.

Imp point:- In 2015, this approach has been used to human cells also.

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