Part:BBa_K2591011
gWls-scaffold
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 this gRNA for wntless WNT ligand secretion mediator (Wls), and it is a functional protein for the secretion of the Wnt3A protein and assist the modification for the mature Wnt3A protein. Therefore, if we transfect the gRNA of Wls 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.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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