We chose CRISPR to knock-out antibiotic resistance. CRISPR system is a bacteria immune system which is used as gene engineer machine now. It has three types and we chose the type II CRISPR system.
This system contains three parts. First, this system can express two kinds of single strand RNA: tracrRNA and crRNA. crRNA’s structure is like “repeat---spacer---repeat”, the spacer part is complementary to target gene’s DNA sequence, the repeat is complementary to tracrRNA while tracrRNA can also have interaction with a protein coded in this system --- Cas9, an endonuclease.
Once we modified the spacer sequence of a CRISPR system and transformed it into bacteria, it will search for target gene (antibiotics resistance gene in our project) in bacteria’s genome by crRNA. Once the target is found, Cas9 protein will bind on this gene’s DNA sequence with the help of tarcrRNA and crRNA. Then, Cas9 will make a small double strand break on the gene. So this gene’s sequence will be modified by later homozygous recombination, result in the knock-out of this gene.
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