The CRISPR-PITCH System Was Used To Construct The B16 Cell Line Expressing CSDE1-EGFP

Objective:B16 melanoma cell line stably expressing CSDE1-EGFP was constructed by using a novel CRISPR-PITCH(Clustered regularly interspaced short palindromic repeats-Precise integration into target chrome)system.According to the fluorescence intensity of EGFP,low expression of cold shock domain with E1(csde1)and high expression of cold shock domain with E1(B16)cell lines were selected by flow cytometry,and their functions were discussed.At the same time,through the establishment of this new CRISPR-PITCH dual system knock-in technology,we can provide technical support for the development of new drug targets.Method:The enhanced green fluorescent protein(EGFP)coding sequence was inserted into the last exon of csde1 genome by CRISPR-PITCH system;Flow cytometry was used to separate and extract genomic DNA,and PCR genotyping was used to detect whether CSDE1-EGFP fusion protein was successfully inserted;Western blotting(WB)and real-time PCR(qPCR)were used to observe the difference of CSDE1 expression between the two cell lines;Live cell imaging and immunofluorescence staining(IF)were used to test whether the fluorescence intensity of EGFP was consistent with that of CSDE1;Transwell test,mouse tumor bearing test and lung metastasis test were used to observe the functional difference between B16 cell lines with low expression of CSDE1 and high expression of CSDE1.Result:The coding sequence of EGFP was successfully inserted into CSDE1 gene by microhomology mediated end joining(MMEJ).The B16 cell lines with low expression of CSDE1 and high expression of CSDE1 were selected by flow cytometry.The expression of EGFP was consistent with that CSDEl.WB,qPCR results verified that there are differences between high and low expression of CSDE1;The results showed that the expression of csde1 protein was different in the function of melanoma cells.Compared with B16 cells with low expression of csdel,B16 cells with high expression of csde1 had stronger metastasis,invasion and tumor forming ability.Conclusion:The new CRISPR-PITCH system gene knock-in technology provides a fast and effective method to study the dynamic localization of CSDE1 in other tumor cells and the functional differences caused by different expression levels.