Hi
I have a set of three gRNAs targeting the promoter region of a specific gene of interest, the issue is that all three gRNAs work in vitro however are in effective in vivo in HEK293 cells and CFBE41o cells (no T7E1 cleavage was detectable) after both lipofection and nucleofection. I have been able to gene edit other genes in these cells just not the promoter region of this specific gene. Therefore I would like to know if anyone has had success gene editing promoter regions of genes? I suspect that the highly conserved nature of promoter sequences and hence the fact that many promoters share conserved sequences might be an obstacle to the gRNA specificity in targeting this region of the genome.
For sure it is very hard to find specific guides in those regions, I asked myself the same question few month ago... I'll stay here to follow the discussion.
Please blast the gRNA sequence and check the specificity. I would recommend using the IDT gRNA designing tool. If the aim of the experiment is to target a specific region in the promoter and your target sequence is highly conserved, then it is actually difficult. But, you also mentioned that the gRNAs works in vitro. Then it should possibly work in HEK and CFBE41o. check the primer specificity for the T7E1 assay.
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