Part:BBa_K2184032

usage and biology

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. 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.

pic 1: Bitter taste perception prevents humans and other mammals from ingesting toxic substances. The perception stems from the binding of bitter molecules to ,25 specific G protein-coupled receptors (GPCRs) referred to as taste 2 receptors (TAS2Rs) TAS2Rs are located in special subsets of taste receptor cells. They are able to detect multiple and diverse natural and synthetic organic molecules.

2. This molecule was specially designed to be able to identify SNPs in grapefroit flavor and mediate CRISPR editing of the non-taste allele.

3. 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.

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.

pic 3:The G protein-coupled receptor (GPCR) TAS2R38 is a bitter taste receptor that can respond to bitter compounds such as phenylthiocarbamide (PTC). This receptor was chosen because its four haplotypes (based on three residue site polymorphism) hTAS2R38PAV, hTAS2R38AVI, hTAS2R38AAI, and hTAS2R38PVV are known to have dramatically different responses to PTC

Functional Parameters

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

generate a synthetic DNA harboring the wildtype (taste) and mutant (non-taste) alleles (SNP) for the ability to sense taste which will be used as control for the Taqman assays and for preforming CRISPR on the mutant SNP allele, to convert it to the wildtype allele, as a feasibility test to be able to change people's taste sense and help them to consume necessary oral medications even if they taste very bitter to them.

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.

References:

Blakeslee, A.F. (1932). Genetics of Sensory Thresholds: Taste for Phenyl Thio Carbamide. Proc. Natl. Acad. Sci. U.S.A. 18:120-130. Fox, A.L. (1932). The Relationship Between Chemical Constitution and Taste. Proc. Natl. Acad. Sci. U.S.A. 18:115-120. Kim, U., Jorgenson, E., Coon, H., Leppert, M., Risch, N., and Drayna, D. (2003). Positional Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to Phenylthiocarbamide. Science 299:1221-1225. Mueller, K.L., Hoon, M.A., Erlenbach, I., Chandrashekar, J., Zuker, C.S., and Ryba, N.J.P. (2005). The Receptors and Coding Logic for Bitter Taste. Nature 434:225-229. Scott, K. (2004). The Sweet and the Bitter of Mammalian Taste. Current Opin. Neurobiol. 14:423-427