CRISPR-Cas9 / insert a gene in cell line genome - (Dec/08/2016 )
Has anyone used CRISPR-Cas9 to insert a gene into the genome of a cell line? I would highly appreciate if you could shoot me with a protocol for that... Thanks so much!!:)
(In more details: I wanted to functionally characterize mutations of a gene, and infect cells with a virus carrying either the wild type or the mutant form of the gene under a CMV promoter. However, the mutant forms turned out to be so damaging for HEK293 cells that they hindered the amplification of the virus itself.
Hence I tried to design a system where I can regulate the expression of my gene of interest as opposed to constitutive expression, and I have designed/preformed viral co-infections where one virus carried a transactivator gene expressed from a CMV promoter, and another virus that carried my gene of interest under a specific regulatory promoter. This regulator promoter lacks binding sites for endogenous mammalian transcription factors, and the only protein that can activate the expression of my gene of interest is the transactivator, translated from the other virus.
The issue was that these two viruses competed with each other for receptor binding sites on the cells, so the expression of my gene of interest was not that great… To overcome this difficulty, I would like to insert the transactivator gene into the genome of the cell line I am working with, so that I only have to infect the cells with one virus that contains my gene of interest under this specific regulator promoter.)
You may want to look into mammalian promoters rather than viral, especially if you want lower expression. There are a bunch of mouse and human ones available.