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.
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.
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
· Escherichia coli
For more bacteria species, please consult us.
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