CRISPR-Cas9 targeted and knockout SLC30A1 gene and specific sgRNA of gene
A specific, MDA-MB-231 technology, applied in the field of sgRNA that specifically targets the SLC30A1 gene, can solve problems such as zinc toxicity
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[0043]1. Construction of a knockout SLC30A1 plasmid using CRISPR / Cas9 technology
[0044] 1 .1 sgRNA oligonucleotide chain synthesis.
[0045] Using the CRISPR online design tool (http: / / crispr.mit.edu / ) according to the scoring system, design a 20bp sgRNA on exon 2 of SLC30A1, and verify that there is no non-specific gene by BLAST. Add CACC to the 5' end of the coding strand template, and add AAAC to the 3' end of the non-coding strand template to complement the cohesive ends formed after digestion with BsmBI, and design a pair of CRISPR oligonucleotide chains, see Table 1 SLC30A1 targeting sites and sgRNA oligonucleotide sequence
[0046]
[0047] 1.2 Vector construction
[0048] 1.2.1 Use BsmBI to digest 2 μg lentiCRISPRv2 plasmid (purchased from Addgene), 2h, 37°C.
[0049]
[0050] 1.2.2 Use the GENRY Gel Recovery Kit to purify the digested plasmid product and operate according to the instructions
[0051] 1.2.3 Phosphorylation and annealing of sgRNA oligos:
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