半岛bd体育手机客户端 说明
一般描述
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 inE. 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 fromStreptococcus 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.
应用
Bacterial Genome Editing
特点和优势
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
原理
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 bacteriumStreptococcus pyogeneshas 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 fromS.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
法律信息
CRISPR Use License Agreement
半岛bd体育手机客户端 性质
| 包装 | vial of 50 μL |
| 浓度 | 20 ng/μL in TE buffer; DNA (1μg of purified plasmid DNA) |
| technique(s) | microbiological culture: suitable |
| 启动子 | Promoter name: AraBAD Promoter activity: inducible |
| application(s) | CRISPR genome editing |
| 运输 | dry ice |
| 储存温度 | ?20℃ |
安全信息
| 储存分类代码 | 12 - Non Combustible Liquids |
| WGK | WGK 2 |
| 闪点(F) | Not applicable |
| 闪点(C) | Not applicable |
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