关键词: CRISPR/Cas9, AAVS1 安全港, 同源重组, SPARK荧光探针, 稳定细胞株, KYSE-150细胞, 细胞外调节蛋白激酶, 表皮生长因子

Abstract:

This work is aimed to construct a KYSE-150 cell line containing an inducible expressed ERK-SPARK kinase fluorescent sensor into the AAVS1 locus. The homologous recombination donor plasmid of the inducible ERK-SPARK expression cassette with 750 bp homology arms at both ends was constructed according to the DNA sequence design of the AAVS1 locus and the published base sequences of the ERK-SPAEK fluorescent probe. Secondly, three sgRNAs targeting the AAVS1 locus were designed using an online design tool, and cloned into the pX330 backbone plasmid. Three CRISPR/Cas9 targeting vectors capable of site-specific cutting of the AAVS1 locus were obtained. The homologous recombination donor plasmids and the expressed CRISPR/Cas9 targeting vectors were co-transfected into KYSE-150 cells, after Dox-induced expression for 24 h, puromycin resistance screening and flow cytometry were performed, and cell subpopulations resistant to puromycin and expressing GFP were obtained. Then PCR was used to identify the genotype, and Western blot was used to detect protein expression. Genomic DNA was extracted from the obtained cell population and the genotype was identified by PCR. The results showed that the size of the obtained fragment was consistent with the size of the inducible ERK-SPARK expression cassette. The results of protein expression detection showed that the cell population expressed the ERK-SPARK probe fusion protein. The results of fluorescent probe imaging experiments demonstrated that the cell line had a very small amount of leakage expression under the condition of no Dox-induced expression. After Dox-induced expression, the GFP fluorescent protein was evenly distributed in the cytoplasm without obvious aggregation into spots. After adding EGF to activate ERK kinase, the green fluorescence of GFP that was uniformly distributed in the cytoplasm gathered into high-brightness green droplets. A stable cell line that expressed the SPARK fluorescent probe after induction for detecting ERK kinase activity were successfully obtained, which may solve the problem that the unstable efficiency of the transiently expressed ERK-SPARK probe affected the accuracy of the experimental results, and may lay the preliminary research foundation for subsequent establishment of screening platform of a SPARK-based high-throughput protein kinase inhibitors.

Key words: CRISPR/Cas9, AAVS1 safe harbor, homologous recombination, SPARK fluorescent sensor, stable cell lines, KYSE-150 cells, ERK, EGF