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