Specifications

Our proposal is to provide a bacterial genome editing approach based on BioBrick parts assembly easy to engineer as A, B, C… CRISPR

The 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 scientific revolution.

In prokaryotes, the genome editing based on CRISPR/Cas9 machinery require quite laborious days of work, the system is weakly founded on bioengineering principles, demands lots of steps, each one carrying a part of the CRISPR/Cas9 apparatus. In addition, off-target events happen frequently, the machinery post-effect in the cell isn’t entirely known yet, among others. To legitimate this information, we integrated our project design with other iGEM's teams who had worked with CRISPR/Cas9 in previous years. You can read more about what we found here

The decoupling of all these shortcomings paved the way to the project's delineation and we elected to focus on a challenge: the lack of standardization, which is a real-world problem - included but not limited to SynBio. Then, to optimize the previous methods, we designed a trustworthy, well functioning standardized framework based on BioBrick parts called: the CRISPeasy toolbox.

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.