CRISPR Non-Target Negative Control Plasmid for Bacteria

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 λ-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 negative 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 Non-Target Negative Control Plasmid for Bacteria (CRISPR31) contains a non-targeting spacer 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.

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 lines

Metabolic Engineering
Strain Optimization

Efficient: increased efficiency of HR-mediated integration
Markerless: does not require antibiotic resistance marker insertion
Scarless: no scar sequences from marker excision which often cause off-target recombination
Multiplexing: multiple custom gRNA sequences can be used at a time

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a single or dual gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Nuclease-based methods are largely toxic when employed as microbial gene editing tools because many bacteria lack the necessary DNA repair mechanisms found in eukaryotic systems. However, when CRISPR/Cas9 is used to mediate recombineering, this cytotoxic quality offers an advantage in that Cas9-induced double stranded breaks kill cells that do not recombine with the donor DNA. This provides an inherent method of selection for markerless, scarless gene editing that is dramatically more efficient and more amenable to multiplexing than traditional methods. The E. coli HR negative control plasmid (Catalog Number CRISPR31-1UG) contains a gRNA sequence targeting no known genomic DNA seqeuence in Wild-type E. Coli. This makes it suitable for use a negative control when used in conjunction with CAS9BAC1P-1UG.