The CRISPR-Mediated Gene Disruption of the Metal-Responsive Transcription Factor-1 (MTF-1) For the Purpose of Creating a Human Knockout Cell Line

The metal-responsive transcription factor-1 (MTF-1) is responsible for regulating the transcription of a cohort of genes, most prominently those involved in the regulation of heavy metal and oxidative stresses. Normally found in both the nucleus and cytoplasm, when MTF-1 encounters excess zinc, either directly through free zinc ions, or indirectly, through its cadmium- or peroxide-triggered release from metallothioneins, it rapidly localizes to the nucleus, where it binds to the promoters of its target genes at conserved “metal-responsive elements.” From there, it can recruit coactivators and general transcriptional machinery to positively regulate gene expression. MTF-1 and several of its target genes have been shown in previous literature to be irregularly expressed in cancer. Our objective in this experiment was to utilize the CRISPR method of gene editing to create a human MTF-1 knockout cell line, so that cancer-associated mutations of MTF-1 can be studied further. The goal of targeting the MTF-1 has been realized. HEK293 was transfected with four guide RNA plasmids for targets located on the first exon of MTF-1, with the goal of generating a premature stop codon that would eliminate translation of the essential nuclear import, DNA-binding, and transactivation domains of MTF-1. T7 Endonuclease assay data demonstrates that transfection and targeting were both highly efficient, with nearly fifty percent of the cell culture possessing a lesion at the CRISPR targets.