Difference between revisions of "Team:Amazonas Brazil/Project"
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In the single guide RNA presence, it forms a catalytically active ribonucleoprotein (RNP) complex | 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 | which cleaves specifics target sequences from DNA, leading to double-strand breaks. Through cell repair | ||
| − | pathways, it will lays the road to genomic editing in living cells. | + | pathways, it will lays the road to genomic editing in living cells. |
| + | |||
| + | Optimized sgRNA (BBa_K2457002): → aqui, estrutura secundária fofinha do sgRNA | ||
| + | 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 | ||
| + | |||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 03:28, 1 November 2017
Navigate into CRISPeasy roadmap
Specifications
Our proposal is to provide a bacterial genome editing approach based on BioBrick parts assembly easy to engineer as A, B, C… CRISPR
Scientific community had achieved great results using CRISPR-Cas9 mediated genome editing techniques. While these methods are moving forward, some basic methodological aspects related to this approach could be further improved to expand this science revolution.
Design
Check all the parts involved and our approaches with closer details!
Cas9 (BBa_K2457001): Standardized Cas9 device, regulated by the 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 lays the road to genomic editing in living cells. Optimized sgRNA (BBa_K2457002): → aqui, estrutura secundária fofinha do sgRNA 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
