Lactobacillus paracasei gene editing system based on endogenous CRISPR-Cas system and application

• Main Authors: ZHANG SHUWEN, PANG XIAOYANG, ZHU QING, XIE NING, LYU JIAPING, XU CHEN, WANG YUNNA
• Format: Patent
• Language: Chinese; English
• Published: 23.06.2023
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

The invention discloses a lactobacillus paracasei gene editing system based on an endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, the gene editing system is characterized in that a mini-CRISPR array expression cassette is inserted into a shuttle plasmid vector pTRKH2, and the array expression cassette comprises a promoter, a crRNA insertion site base sequence with two Bsa1 enzyme cutting sites, an LpCas9SgRNAScaffold base sequence and a terminator. The invention further discloses application of the lactobacillus paracasei gene editing system based on the endogenous CRISPR-Cas system. The targeting vector is free of Cas nuclease, the size of the vector can be greatly reduced, the vector can be converted into lactic acid bacteria more easily, cytotoxicity is avoided, long fragments larger than 1.0 kb can be knocked out, operation is easy, and the period is short. 本发明公开了一种基于内源CRISPR-Cas系统的副干酪乳杆菌基因编辑系统，该基因编辑系统包括：通过在穿梭质粒载体pTRKH2上插入mini-CRISPR阵列表达框，阵列表达框包括：启动子、带有两个Bsa1酶切位点的