Part:BBa_K2184002

Guid RNA for Cas9 for coffee biterness flavor

1. The basic assumption of the project is based on the fact that there is a direct link between particular taste affection and the number of receptors for that taste. For this reason, the ability to control the polymorphism of taste receptors may be useful in many ways to human society

2. Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule.

3. This molecule was specially designed to be able to identify SNPs in coffee biterness flavor and mediate CRISPR editing of the non-taste allele only.

Use and Biology

Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. The basic assumption of the project is based on the fact that there is a direct link between particular taste affection and the number of receptors for that taste. For this reason, the ability to control the polymorphism of taste receptors may be useful in many ways to human society, for example, specific desirable diet aid by reducing the ability to feel a specific unhealthy taste, reducing alcohol consumption by changing sensitivity to alcohol, reducing the risk of high blood pressure by eliminating the need for salt taste, changing the sensitivity for bitterness to allow swallowing a pill for the purpose of providing medical care and more.

Functional Parameters

This part was used and validated by danci k8 team in a following construct:

1. Guide RNA consists of a unique two dimensional structure(crRNA and tracrRNA) which binds Cas9 and guides it to a dsDNA sequence complementary to the 5' end of the molecule.

2. Identification of the selected allele for a specific taste receptor encoded and sliced in vitro using the Cas9 enzyme. This change will allow us to control and navigate the ability of the sense of taste as needed.

3. Five synthetic gRNA (guide RNA) molecules- These molecules were specially designed so that four of them (gRNA No. 2-5) were able to identify SNPs in four flavors respectively and mediate CRISPR editing of the non-taste alleles only, gRNA No. 1- molecule does not identify a specific SNP, and will be used as negative control.

4. Cas9 endonuclease - Cas9 is the endonuclease guided by the crRNA and tracrRNA (or trans-activating crRNA) to cleave specific DNA sequences. Binding specificity is based on the gRNA sequence and a three nucleotide NGG sequence called the protospacer adjacent motif (PAM) sequence.