Abstract: Objective To construct a melanoma cell strain expressing endotopic CSDE1-EGFP using new CRISPR-PITCH system, then to select the cell strain of low expression CSDE1 and high expression CSDE1. Methods The EGFP coding sequence was inserted into the last exon of the CSDE1 gene using a micro-homology-mediated end joining(MMEJ) method; Using flow cytometry sorting, PCR, immunofluorescence and live cell imaging to detect whether CSDE1-EGFP was inserted successfully, and whether the fluorescence intensity of EGFP was consistent with the expression level of CSDE1; The Transwell experiment and mouse tumor-bearing experiment were used to observe the functional differences between the B16 cell strain with low and high expression of CSDE1. Results The B16 cell strains of low expression CSDE1 and high expression CSDE1 were selected, and the strength of EGFP expression was found to be consistent with CSDE1 expression. The melanoma cells with different expression level of CSDE1 protein had different functions. Conclusions The new CRISPR-PITCH system provides a fast and effective intuitive method for studying the dynamic positioning of CSDE1 in other tumor cells and the functional differences are resulted from different expressions.

Key words: CRISPR-PITCH system, CSDE1, micro-homology-meditated end joining(MMEJ), enhanced green fluorescence protein(EGFP)