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.

guide RNA (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 (Bba_K2457004) This BioBrick contains homologous arms (300bp upstream and downstream from the Operon Lac, to increase the efficiency) as a template to insert information into genome on the repair pathway of cells. Allows the recombination through the RecA activity by Homology Directed Repair (HDR) pathway. It has in its coding sequence the Green Fluorescent Protein (GFP) gene which will operate as a reporter to recombinant cells that will become fluorescents.