Provided are CRISPR/Cas9 system-based directed RNA(gRNA) sequences and combinations thereof in preparation of gRNA for specifically targeted knockout of the human PDCD1L1 gene. The gRNA is designed according to a CRISPR/Cas9 design principle, the sequence of the gRNA is as shown in SEQ ID NO.1, and the gRNA is constructed on a px459 vector. In Jurkat cells, the CRISPR/Cas9 system directed by the gRNA and combination thereof can effectively knock out the human PDCD1L1 gene. The prepared gRNA can accurately target the human PDCD1L1 gene and implement a gene knockout. The preparation method is simple in operation, the gRNA is good in targeting, and knockout efficiency of the CRISPR/Cas9 system is high.
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