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1. (WO2018182361) METHOD FOR PREPARING CORYNEBACTERIUM MUTANT STRAIN, USING CRISPR/CAS SYSTEM, RECOMBINASE, AND SINGLE-STRANDED OLIGODEOXYRIBONUCLEIC ACID

Pub. No.:    WO/2018/182361    International Application No.:    PCT/KR2018/003784
Publication Date: Fri Oct 05 01:59:59 CEST 2018 International Filing Date: Sat Mar 31 01:59:59 CEST 2018
IPC: C12N 15/77
C12N 15/64
C12N 15/65
C12N 15/113
C12P 13/00
Applicants: KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY
한국과학기술원
Inventors: LEE, Sang Yup
이상엽
CHO, Jae Sung
조재성
CHOI, Kyeong Rok
최경록
Title: METHOD FOR PREPARING CORYNEBACTERIUM MUTANT STRAIN, USING CRISPR/CAS SYSTEM, RECOMBINASE, AND SINGLE-STRANDED OLIGODEOXYRIBONUCLEIC ACID
Abstract:
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.