This product is not tested for the specific application. However, if the phage genome has been integrated into the E. coli genome, researcher could target this phage DNA sequence for integrating a gene. Please note that the process will not work if targeting just independent phage particles.
CAS9BAC1P
Cas9 Lambda Red Homologous Recombination Plasmid for E. Coli
Synonym(s):
Cas9 plasmid, Lambda Red plasmid
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| Pack Size | SKU | Availability | Price |
|---|---|---|---|
| 1 ea | Usually ships in 3 weeks. | $279.00 |
About This Item
NACRES:
NA.51
UNSPSC Code:
41106617
Promoter:
Promoter name: AraBAD
Promoter activity: inducible
Promoter activity: inducible
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%[1], 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[2].
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
Biochem/physiol Actions
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
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
Legal Information
1 of 1
This Item | |||
|---|---|---|---|
| promoter Promoter name: AraBAD | promoter
| promoter
| promoter - |
| form liquid | form liquid | form liquid | form - |
| storage temp. −20°C | storage temp. −20°C | storage temp. −20°C | storage temp. −70°C |
| technique(s) microbiological culture: suitable | technique(s) microbiological culture: suitable | technique(s) microbiological culture: suitable | technique(s) - |
| shipped in dry ice | shipped in dry ice | shipped in dry ice | shipped in dry ice |
| application(s) CRISPR | application(s) CRISPR | application(s) CRISPR | application(s) - |
Storage Class
12 - Non Combustible Liquids
wgk
WGK 2
flash_point_f
Not applicable
flash_point_c
Not applicable
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Articles
E. coli CRISPR/Cas-mediated Lambda-Red HR vector system enables gene editing via homology-directed repair.
Related Content
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
Global Trade Item Number
| SKU | GTIN |
|---|---|
| CAS9BAC1P-1EA | 04061841332442 |
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Can i use this system for integrating a gene into an E. coli Phage genome like T4 or T7?
1 answer-
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I need to delete a gene in 2 different types of E. coli. Can I do it by multiplex primer design ?
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In theory, this process is possible. However, it would be best to do one mutation at a time due to the screening challenges for multiple mutations.
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Is there a restriction site on the plasmid for linearization? I want to check the presence on the gel.
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As shown in the plasmid image located on the Product Detail Page, it does not have a restriction site.
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What gene is responsible for the KanR in the CAS9BAC1P (Cas9 Lambda Red Homologous Recombination Plasmid)?
1 answer-
The gene responsible for the KanR in CAS9BAC1P is Serratia marcescens.
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