Patent ID: 11911455
Assignee: BEIJING AEONVITAL BIOMEDICINE RESEARCH CO., LTD.
Field: Pharmaceuticals (Chemistry)
Classification: CPC A  C  Y | IPC A  C

Claim 15:
16. The preparation method according to claim 15, characterized in that: the gene editing is achieved by a method comprising the following steps:
i) PCR amplification of the upstream and downstream homologous arm gene sequences of the insertion site, p5/6 6/6-SacB gene sequence respectively, and overlapping extension PCR with the three sequences as templates to amplify the long fragment SacB-CRISPR nucleotide sequence; the nucleotide sequence of SacB-CRISPR is shown as SEQ ID No: 18;
ii) Co-transforming the Cas9 expression plasmid, sgRNA, and the long fragment SacB-CRISPR nucleotide sequence into Escherichia coli DH5a competent cells, performing gene editing and homologous recombination repair, selecting single clones for culture, and verifying by PCR sequencing; finally, eliminating the resistance of tool plasmid and the edited strain by the temperature-sensitivity to obtain the AVL-DH5a strain suitable for target plasmid without antibiotics selection.