The essential PAM sequence for gene editing was selected, and target sites were designed using the online CRISPR target site design tool E-CRISPR http://www.e-crisp.org/ (accessed on 21 December 2019), for the construction of a single-target knockout vector. The primers for target site design are listed in Table 1, and were synthesized by Tsingke Biotechnology (Guangzhou, China) Co., Ltd. The constructed vector was sent to Biorun Biosciences Co., Ltd. (Wuhan, China).|||PCR products ware ligated into the T19 simple vector, and transformed into Escherichia coli DH5α. Ten independent monoclonal colonies were selected and sent to Tsingke Biotechnology (Shanghai, China) Co., Ltd. for duplex sequencing. The sequencing results of mutants and wild type (WT) were aligned using SnapGene software 3.2.1.
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