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CRISPR30

Sigma-Aldrich

CRISPR LacZ Positive Control plasmid for Bacteria

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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 activity: constitutive

shipped in

dry ice

storage temp.

−20°C

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CRISPR31CAS9BAC1PCRISPRPL
vibrant-m

CRISPRPL

CRISPR Plant

form

liquid

form

liquid

form

liquid

form

-

shipped in

dry ice

shipped in

dry ice

shipped in

dry ice

shipped in

dry ice

technique(s)

microbiological culture: suitable

technique(s)

microbiological culture: suitable

technique(s)

microbiological culture: suitable

technique(s)

-

storage temp.

−20°C

storage temp.

−20°C

storage temp.

−20°C

storage temp.

−20°C

packaging

vial of 50 μL

packaging

vial of 50 μL

packaging

vial of 50 μL

packaging

-

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 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.

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 lines
Metabolic Engineering
Strain Optimization

Features and Benefits

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

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 Positive Control Plasmid (Catalog Number CRISPR30) contains a gRNA sequence targeting the lacZ gene in wild-type E. coli. and is designed to be used in conjunction with the E. coli HR Cas9 Plasmid (Catalog Number CAS9BAC1P) and a ssDNA donor template.

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


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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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