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<h2 class="Page_Subtitle" align="middle">pCRISPeasy</h2> | <h2 class="Page_Subtitle" align="middle">pCRISPeasy</h2> | ||
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− | <img alt="Erro" style="width: 80%;height: 80%;margin-left: 10%" | + | <img alt="Erro" style="width: 80%;height: 80%;margin-left: 10%" |
src="https://static.igem.org/mediawiki/2017/f/fa/Amazonas_Brazil_Project_Specifications.png"> | src="https://static.igem.org/mediawiki/2017/f/fa/Amazonas_Brazil_Project_Specifications.png"> | ||
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Revision as of 08:48, 1 November 2017
Navigate into CRISPeasy roadmap
![Erro](https://static.igem.org/mediawiki/2017/f/fa/Amazonas_Brazil_Project_Specifications.png)
![Erro](https://static.igem.org/mediawiki/2017/1/14/Amazonas_Brazil_Project_Prokaryotes.png)
![Erro](https://static.igem.org/mediawiki/2017/3/3f/Amazonas_Brazil_Project_Events.png)
![Erro](https://static.igem.org/mediawiki/2017/5/5b/Amazonas_Brazil_Project_Lack.png)
Design
Check all the parts involved and our approaches with closer details!
Cas9 (BBa_K2457001)
The standardized Cas9 device under control of AraC_pBAD regulatory module. In the single guide RNA presence, it forms a catalytically active ribonucleoprotein (RNP) complex which cleaves specifics target sequences from DNA, leading to double-strand breaks. Through cell repair pathways, it will lay the road to genomic editing in living cells.
Optimized sgRNA (BBa_K2457002)
This biobrick is constitutively expressed by J23100 promoter and codify an optimized sgRNA chimera composed of a protospacer crRNA with a GG motif at the 3’ end of its target-specific sequence, a tracrRNA with extended duplex length and a mutation at the thymine 4. It was rationally engineered to improve the editing efficiency of CRISPR-Cas9 machinery. When expressed, it guides the Cas9 nuclease activity through Rosalind-Watson-Crick base pairing complementarity with the target sequence.
RecA (Bba_K2457003)
RecA is an essential building block part which leads the homologous recombination AND DNA insertion into the genome. After the target sequence cleavage catalyzed by the Cas9-sgRNA complex, the SOS response machinery is activated. As an output of the repair pathway, RecA builds a DNA-protein filament which investigates for a homologous template on undamaged DNA sequences, assisting in the invasion process and setting-up the Holliday Junction. This generates a crossover with the strands, leading to the recombination among the undamaged strand and the broken strand.
DONOR DNA![](https://static.igem.org/mediawiki/2017/5/5f/T--Amazonas_Brazil--infografico_project.png)