CRISPR/Cas Method of Preparing Corynebacterium Variant Based on CRISPR/Cas System Recombinase and ssODN

摘要

The present invention comprises the steps of (a) transforming normal bacteria into temperature-sensitive or antibiotic-sensitive, (i) primary transforming with an enzyme expression vector expressing a recombinant enzyme (recombinase); (b) preparing a competent cell of the primary transformed bacteria obtained in step (a); (c) (i) a single-stranded oligodeoxyribonucleic acid (ssODN) and (ii) a guide complementarily binding to the target gene to the recipient cell obtained in step (b) Second transforming by introducing a first vector expressing RNA (guide RNA) and having antibiotic or temperature sensitivity; And (e) removing the enzyme expression vector and the first vector inserted from the secondary transformed bacteria, wherein at least one of the enzyme expression vector and the first vector expresses Cas protein. It relates to a method of manufacturing a bacterial mutant. The method according to the present invention can effectively delete the target gene, select recombinant microorganisms quickly and with high efficiency, and also remove all foreign vectors in the finally selected recombinant microorganism, so that the recombinant Coryne It is very useful for industrial use of Bacterium.