RNA-Programmed Gene Circuit Engineering Using Crispr-Nickases
Synthetic Biology Engineering Evolution Design SEED
2016
2016 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Accepted Posters
There is a need for programmable tools to remodel genomes. Such tools have been integral for synthetic biologists to engineer organisms and implement synthetic gene circuitry. CRISPR-Cas systems provide an easily programmable tool for targeting many of these precise manipulations. Here we demonstrate the ability of CRISPR-Cas derived nickases to direct targeted-recombination of large genomic regions flanked by repetitive elements in E.coli. While CRISPR directed double-stranded DNA breaks are highly lethal in many bacteria, we show that CRISPR-guided nickase systems can be programmed to make precise, non-lethal, single-stranded incisions in targeted genomic regions. We demonstrate that dual-targeted nicking induces recombination events resulting in large-scale deletion. Multiplex targeting enables deletion of 133 Kb, approximately 3% of the entire E.coli genome. This provides a framework for methods to manipulate bacterial genomes using CRISPR-nickase systems. We envision this system working synergistically with preexisting bacterial genome engineering methods with further extension enabling targeted and multiplex integration of synthetic gene circuits. Future development of CRISPR based methods may synergistically incorporate aspects of protein engineering and multi-targeted sgRNA design to accelerate genome engineering.