Cas9 Lambda Red Homologous Recombination Plasmid for E. Coli

Descrição detalhada : Recent publications using CRISPR/Cas9-mediated recombineering in E. coli tout editing efficiencies near 100%, making CRISPR/Cas9-mediated recombineering the most powerful bacterial genome engineering method to date. In addition, Cas9-mediated recombineering overcomes the dependence on a second recombination step, avoids the creation of destabilizing scar sites, can be used in multiplexing, and is less time-consuming than previous protocols.Here we present a novel dual-vector CRISPR/Cas-mediated ?-Red system for improved recombineering in E. coli. Our system is shown to facilitate homology-directed repair of DSBs created by Cas9 endonuclease, enabling genetic alterations through chromosomal integration of a donor DNA.This plasmid is to be used in combination with a custom gRNA (CRISPRBACD) which can be designed and ordered through our Custom gRNA Design Tool. The donor can be either ssDNA or dsDNA with homology arms of 45-59 or 150-500 nucleotides respectively. Protocols for donor design can be found in the technical bulletin.The Cas9 Lambda Red Homologous Recombination Plasmid for E. coli (CAS9BAC1P) contains the gene for Cas9 from Streptococcus pyogenes (spCas9) expressed from its native promoter, as well as the genes for ?-red recombinases exo, beta, and gam under the control of the arabinose-inducible ParaB promoter. This plasmid confers kanamycin resistance and possesses the repA101ts temperature-sensitive origin of replication, allowing for easy plasmid maintenance and curing.
Armazenamento : -20C
Embalagem : 1X1EA