Part:BBa_K2591020
A gRNA designed to PORCN, an essential gene to secretion of the canonical wnt protein
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.
Characterization
Target length: 20 nt
Target sequence: 5’-GTGACATGGCACAAGATGCG-3’
Primers for plasmid construction:
Porcn-gRNA-F: 5’- CACCGTGACATGGCACAAGATGCG-3’
Porcn-gRNA-R: 5’- AAACCGCATCTTGTGCCATGTCAC-3’
Location in gene loci:
As mentioned above, knockout of PORCN will impair the Wnt3a secretion activity in the cells. Therefore, we first introduce sgPor into L-Wnt3A-Cas9-mCherry cell line, which continuously expresses Cas9 protein. And then coculture with our Wnt3a reception cell, 293R-TCF-EGFP cell, then observed the fluorescence after one day to validate our part. Validation images are shown in Fig3.
Figure 4. 293R-TCF-EGFP cell line coculture with the L-Wnt3A-Cas9-mCherry-gRNA_PORCN cell line in 24h. (a) observe with white light; (b) observe at 475nm; (c) observe at 556nm. We cannot see green fluorescent protein at the 475nm, it means the Wnt3A protein cannot be secreted from the L cell normally, and the gRNA designed for PORCN could target specific sequence and mutate it with Cas9 protein.
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]
| None |