Question

Imagine you have discovered a specific mutation (an A in the gene has been mutated to...

Imagine you have discovered a specific mutation (an A in the gene has been mutated to a T) in several patients that causes them to have neon green tongues. You want to study this specific mutation in mice and plan to use the CRISPR/Cas9 system to allow you to do so. How might you go about doing this?

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The Cas9 is known to possess an endo nuclease activity that cuts double stranded DNA. The CRISPR/Cas9 system could be used for cleaving DNA at site and for recognition as well.To make this possible, Cas9 is complexed with the crRNA and trRNA. The trRNA is being used as it is required for the maturation of crRNA. A double stranded break will result as Cas9 cuts double stranded DNA at specific site.We can also proceed with site specific sequencing without cleavage.For this, we can use a nuclease deficient Cas 9 in which the binding activity is retained but cleavage function is deactivated.This system is fused with the EGOF(Enhanced green fluorescent protein) so that the binding can be visualised. This visualisation would give an idea of the repetitive DNA sequences.

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