Method for knocking out PD-1 gene by utilizing CRISPR/Cas9 technology to construct MSLN-targeted novel CAR-T cell and application of method
A PD-1 and gene technology, applied in the fields of genetic engineering, immunology and oncology, can solve the problems of no observed side effects, increased white blood cell level, etc., and achieve the effect of simple steps and high killing rate
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[0050] A method for constructing a novel CAR-T cell targeting MSLN by using CRISPR / Cas9 to specifically knock out the human PD-1 gene, comprising the following steps:
[0051] 1) Construction of viral vectors carrying PD-1 gene sgRNA oligonucleotides
[0052] The target sequence of the sgRNA on the PD-1 gene conforms to the sequence arrangement rules of 5'-N(20)NGG, and the target sequence of the sgRNA on the PD-1 gene is located in the exon of the gene; the target sequence of the sgRNA on the PD-1 gene The target sequence is unique, and the target site sequence of the sgRNA on PD-1 consists of the human PD-1 gene targeting sgRNA sequence PD-1-Tn shown in SEQ ID No.51 to SEQ ID No.56;
[0053] The mRNA sequence of the human PD-1 gene consists of human PD-1 gene exons shown in SEQ ID No.46 to SEQ ID No.50;
[0054] Synthesize sgRNA oligonucleotide double strands (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), anneal at 95°C for 5 minutes after mixing, and then con...
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