Difference between revisions of "Part:BBa K2591010"

Revision as of 17:29, 11 October 2018

gPor-scaffold

Enter a long description of the part so that users of your part know what it is, what it does, and how to use it in their projects.

Usage and Biology

Naturally, single guide RNA(sgRNA) is a small RNA which guide CRISRR-Cas protein family to target the exogenous sequence in prokaryotes, and assists them to defend phages. Nowadays, the artificial CRISPR-Cas9 system can achieve the modification casually in the gene level and is an excellent way to introduce mutation. The mechanism is shown in Fig1, the Cas9 nuclease is targeted to genomic DNA by a sgRNA consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence pairs with the DNA target (blue bar on top strand), directly upstream of a require a 5’-NGG adjacent motif (PAM; pink). Cas9 mediates a double strand break in the upstream of the PAM (red triangle).(Ran, et al, Nat. Protoc., 2013)

Figure1. Schematic of the RNA-guided Cas9 nuclease.(Ran, et al, Nat. Protoc., 2013)

In our project, we design the gRNA for porcupine O-acyltransferase (PORCN), an essential enzyme to function in the Wnt3A protein secretion. Therefore, if we transfect the gRNA of PORCN into cells, we can inhibit the donor cell to secret the Wnt3A protein normally. And we have integrated this gRNA together with its scaffold into the backbone pSB1C3 to get a new part.(Fig2)

Figure2. Diagram of pSB1C3_gRNA_scaffold.

characteriazation

Sequence and Features