Patent ID: 11866703
Assignee: LANZHOU VETERINARY RESEARCH INSTITUTE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 5:
6. The knockout method according to claim 1, further comprising constructing the pCRISPR::EtNMT plasmid before step a, comprising:
i. with a pSAG1::Cas9-U6::sgUPRT plasmid as a template, conducting amplification using a CRISPR-EtNMT-F primer and a CRISPR-R primer to obtain an amplification product; and
ii. subjecting the amplification product to a Kinase, Ligase, and Don1 (KLD) reaction to obtain a reaction product; and
iii. transforming the reaction product into competent cells; and
iv. extracting a resulting plasmid to obtain the pCRISPR::EtNMT plasmid; wherein the CRISPR-EtNMT-F primer has the nucleotide sequence shown in SEQ ID NO: 1; and
the CRISPR-R primer has the nucleotide sequence shown in SEQ ID NO: 2.