Traditional bacterial gene editing mainly uses the RecBCD system that widely exists in bacteria to recombine
the suicide plasmid with the genome to knock out the target gene. However, the efficiency of this method is
extremely low, which requires repeated experiments, and may also lead to reverse mutation, and it is
difficult to obtain the target gene knockout strains. λ-Red is a better choice for Escherichia coli and its
close species. Its recombination efficiency will be slightly higher than the endogenous recombination system
in bacteria, but the target gene needs to be replaced with a resistance gene. After successful replacement,
although the recombinant enzyme can be transferred to bacteria to delete the resistance gene, it will still
leave a recombination site on the genome.
Based on the above problems, the CRISPR-B™ technology system developed by Ubigene innovatively
combines the Red/ET recombination system with CRISPR/Cas9 gene editing system, uses the
advantages of CRISPR/Cas9 system in efficient cutting and further optimizes the gene editing plasmid and
gene editing process, greatly improving the efficiency of bacterial gene editing. Compared with the previous
two methods, the CRISPR-B™ technology of Ubigene can not only achieve high efficiency, but also achieve
scarless knockout (that is, no residual resistant gene or recombination sites, and eliminate foreign
plasmids at the same time). In addition, this technology can achieve target gene point mutation and knockin
which cannot be achieved by the previous two methods. At present, Ubigene can provide gene editing
(knockout/point mutation/knockin) and construction of overexpression strains of Escherichia coli,
Salmonella and Pseudomonas aeruginosa, guaranteeing the delivery of positive homozygous clones.
Escherichia coli, uses gene editing technology to modify its genome to study gene
function, or change its metabolic pathway to produce a large number of commercially valuable
products, so as to obtain genetically stable engineering strains that can produce specific products.
Salmonella is a genus of Enterobacteriaceae, gram-negative. In addition to the study of
pathogens, it can also be used to establish commonly used mouse gastroenteritis models and explore
more genetic tools (for example, the first extensive transduction phage P22 was found in Salmonella
Pseudomonas aeruginosa is a kind of conditional pathogenic bacteria that widely exists
in the environment. It is also an important model organism to study the quorum sensing, biofilm,
virulence and drug resistance of gram-negative bacteria.
Staphylococcus aureus is a representative gram-positive bacteria, a common food-borne pathogenic microorganism, which can cause suppurative infection, pneumonia, enteritis, endocarditis, sepsis, and septicemia. The abuse of antibiotics can lead to the development of resistance in Staphylococcus aureus, and the effectiveness of existing treatment options is becoming increasingly unsatisfactory. Therefore, research on its drug resistance mechanism and new treatment strategies is currently of utmost importance.
Service type: scarless gene knockout, point
mutation, knockin, gene overexpression
Deliverable: Positive clone
The efficiency is 20-30 times higher than that of the classic methods;
Scarless gene-editing technology, safe and sound;
Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI);
It is possible to knockout multiple genes simultaneously.
CRISPR-B™ vector construction
|CRISPR-B™ vector construction |
|CRISPR-B _CR||Carrying Cas9 nuclease and Red recombinase|
|CRISPR-B _G||Carrying donor template|
|CRISPR-B _D||Carrying target gene gRNA|
（2）Electroporation and CRISPR-B™ _CR competent cells preparation
· CRISPR-B _CR vector transfer into bacteria by electroporation.
Validate the transfection by colony PCR and sequencing.
·Select the CRISPR-B _CR positive strains
and prepare the CRISPR-B _CR competent cells
（3）Transfer of CRISPR-B_G and CRISPR-B _D
Transfer vectors CRISPR-B_G and CRISPR-B_D into CRISPR-B _CR competent cells by
electroporation. CRISPR and Red recombination system worked together to
edit the bacterial genome.
（4）Validate the single clones by PCR and sequencing. Eliminate CRISPR-B™ plasmids, and
obtain the knockout clones.
1. Colony PCR 2.Sequencing