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 the Cas9 Lambda Red homologous recombination plasmid for E. coli (CAS9BAC1P) as the positive control for your custom gene editing experiment. The custom gRNA (CRISPRBACD) can be designed and ordered through https://www.sigmaaldrich.com/pc/ui/genomics-home/customcrispr The CRISPR LacZ Positive Control Plasmid for Bacteria (CRISPR30) contains a gRNA spacer targeting the lacZ gene in wild-type E. coli expressed constitutively from a J23119 promoter, a ampicillin resistance marker, a pBR322 origin of replication, and a sacB gene from Bacillus subtilis for counter-selection-based curing.
Storage: -20C
Application: Bacterial Genome Editing
•HR-mediated recombineering for mutation or SNP analysis
•Creation of HR-mediated knock-in cell lines with promoters, fusion tags, or reporters integrated into endogenous genes
•Creation of gene knockouts in E. coli cell linesMetabolic EngineeringStrain Optimization
Legal Information: CRISPR Use License Agreement