One-Step in Vitro Large Gene Cluster Cloning By Crispr-Cas9 with Gibson Assembly
Synthetic Biology Engineering Evolution Design SEED
2016
2016 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Accepted Posters
We describe a method that allows the targeted cloning of near-arbitrary, long genomic sequences (50-150 kb) from bacteria to accomplish in a single step within 1-2 days. Target DNA is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This method can be a useful molecular engineering tool for efficiently cloning large gene clusters that are difficult to obtain directly by traditional PCR or restriction-enzyme-based methods.
