Difference between revisions of "Part:BBa K2591012"
(→Usage and Biology) |
|||
| Line 4: | Line 4: | ||
===Usage and Biology=== | ===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 wingless-type MMTV integration site family, member 3A (Wnt3A). Wnt3A protein is a secretion protein function in the development and proliferation of the normal cell, and more importantly, canonical Wnt pathway play a role in the induction of cancer. In our part, we want to explore the upstream of the Wnt pathway, and want to find the gene factors which affect the Wnt pathway by the high-throughput double emulsion system. Thus, we design this gRNA for Wnt protein coding sequence as a positive control. 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=== | ===Characterization=== | ||
Revision as of 01:22, 12 October 2018
gWnt3a-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 wingless-type MMTV integration site family, member 3A (Wnt3A). Wnt3A protein is a secretion protein function in the development and proliferation of the normal cell, and more importantly, canonical Wnt pathway play a role in the induction of cancer. In our part, we want to explore the upstream of the Wnt pathway, and want to find the gene factors which affect the Wnt pathway by the high-throughput double emulsion system. Thus, we design this gRNA for Wnt protein coding sequence as a positive control. 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]