Abstract: With the CRISPR gene editing technology widely implemented in both plants and animals, a multi-gene editing system in breast cancer was constructed by harnessing the self-shearing characteristics of endogenous tRNA, integrating tRNA and multiple single guided RNA (sgRNA) targeting genes of interest into a single plasmid. Four transcription factors KLF4, MYC, SOX2 and OCT4 were modulated simultaneously using our system in breast cancer cell line MCF7 where the function and reliability of our system was verified from the transcriptional expression, protein expression and functional changes assays. The expressions of all modulated genes were upregulated above 2-fold, with the modulated cell displayed higher migration and invasive abilities and an increased cell availability by 50%. The proportion of stem cells was also increased from 2.57% to 18.5%. Concomitant multiplexing modulation of the same gene panel using our constructed system in breast cancer cell line SUM149PT was also achieved. Thus, based on CRSIPR, a standard, accurate, reproducible and efficient multiplexing gene modulation system in breast cancer cells was successfully built.

Key words: breast cancer, CRISPR, gene reprogramming