Search International and National Patent Collections
|1. (WO2018182361) METHOD FOR PREPARING CORYNEBACTERIUM MUTANT STRAIN, USING CRISPR/CAS SYSTEM, RECOMBINASE, AND SINGLE-STRANDED OLIGODEOXYRIBONUCLEIC ACID|
|Applicants:||KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY
|Inventors:||LEE, Sang Yup
CHO, Jae Sung
CHOI, Kyeong Rok
|Title:||METHOD FOR PREPARING CORYNEBACTERIUM MUTANT STRAIN, USING CRISPR/CAS SYSTEM, RECOMBINASE, AND SINGLE-STRANDED OLIGODEOXYRIBONUCLEIC ACID|
The present invention relates to a method for preparing a bacteria mutant strain, comprising the steps of: (a) primarily transforming a normal bacteria into an enzyme-expressing vector which has temperature sensitivity or antibiotic sensitivity and expresses (i) a recombinase; (b) making the primarily transformed bacteria obtained in step (a) to be a competent cell; (c) secondarily transforming the competent cell obtained in step (b) by introducing (i) a single-stranded oligodeoxyribonucleic acid (ssODN) which binds complementarily to the target gene and (ii) a first vector which expresses a guide (RNA) and has antibiotic sensitivity or temperature sensitivity; and (e) removing the inserted enzyme-expressing vector and the first vector from the secondarily transformed bacteria, wherein at least one of the enzyme-expressing vector and the first vector expresses a Cas protein. A method according to the present invention can delete a target gene effectively, select a recombinant microorganism rapidly and at high efficiency, and remove all foreign vectors from the finally selected recombinant microorganism, finding very useful applications in the industrial use of recombinant Corynebacterium.