CAS9BAC1P
Cas9 Lambda Red Homologous Recombination Plasmid for E. Coli
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About This Item
UNSPSC Code:
41106617
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form
liquid
packaging
vial of 50 μL
concentration
20 ng/μL in TE buffer; DNA (1μg of purified plasmid DNA)
technique(s)
microbiological culture: suitable
application(s)
CRISPR
genome editing
Promoter
Promoter name: AraBAD
Promoter activity: inducible
shipped in
dry ice
storage temp.
−20°C
General description
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 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 λ-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.
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 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 λ-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.
Application
Bacterial Genome Editing
Strain Optimization
- 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
Strain Optimization
Features and Benefits
Efficient: increased efficiency of HR-mediated integration to almost 100%
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
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
Principle
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 Cas9 Plasmid (Catalog Number CAS9BAC1P) contains the gene for Cas9 from S.pyogenes (spCas9) expressed from its native promoter, as well as the genes for λ-red recombinases exo, beta, and gam under the control of the arabinose-inducible ParaB promoter. This plasmid confers ampicillin resistance and possesses the repA101ts temperature-sensitive origin of replication, allowing for easy plasmid maintenance and curing
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Description
Pricing
Storage Class Code
12 - Non Combustible Liquids
WGK
WGK 2
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Certificates of Analysis (COA)
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Yifan Li et al.
Metabolic engineering, 31, 13-21 (2015-07-05)
Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli.
Michael E Pyne et al.
Applied and environmental microbiology, 81(15), 5103-5114 (2015-05-24)
To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex
Articles
In this article, we present an application of our novel E. coli CRISPR/Cas-mediated Lambda-Red (λ-Red) homologous recombination (HR) vector system, which facilitates gene editing through the homology-directed repair (HDR) of double-stranded DNA breaks (DSBs) created by Cas9 endonuclease, using either ssDNA or dsDNA as an editing template.
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