Gene-editing Microorganisms
Red/ET recombination system is a classical method of microbial gene editing, which can achieve the knockin, knockout, point mutation and other modifications of the target gene. This technology has been widely used in genetic modification of genomic DNA, such as bacterial artificial chromosome(BAC), Escherichia coli chromosome. However, the efficiency of this system still needs to be improved.
How to improve the efficiency of gene recombination and editing has always been a hotspot of microbial gene editing. Therefore, CRISPR/Cas9 technology is adopted to improve the efficiency of microbial gene editing.
CRISPR/Cas9 is an acquired immune system in bacteria and archaea and can be used to fight against invading viruses and exogenous DNA. In recent years, the CRISPR/Cas9 gene-editing technology has been widely used because it is simple and efficient. It has been the most advanced method for gene editing.
Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi.
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Red/ET recombination system is a classical method of microbial gene editing, which can achieve the knockin, knockout, point mutation and other modifications of the target gene. This technology has been widely used in genetic modification of genomic DNA, such as bacterial artificial chromosome(BAC), Escherichia coli chromosome. However, the efficiency of this system still needs to be improved.
How to improve the efficiency of gene recombination and editing has always been a hotspot of microbial gene editing. Therefore, CRISPR/Cas9 technology is adopted to improve the efficiency of microbial gene editing.
Technical advantages
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.
Editing bacteria
Ubigene uses CRISPR-B™ system to modify the genome of fungi and achieve knockout, point mutation or knockin. Customers could choose the genome editing methods with or without residual.
What bacteria can CRISPR-B™ modify?
Some gram-positive bacteria
· Bacillus subtilis
· Streptococcus thermophilus
Most of the gram-negative bacteria
· Escherichia coli
· Salmonella
· Pseudomonas aeruginosa
For more bacteria species, please consult us.
Work Flow
CRISPR-B™ vector construction
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CRISPR-B™ vector construction
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CRISPR-B _CR
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Carrying Cas9 nuclease and Red recombinase
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CRISPR-B _G
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Carrying donor template
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CRISPR-B _D
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Carrying target gene gRNA
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（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.
Validation
1. Colony PCR 2.Sequencing
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