Hi,
Many people uses CRISPR Homology repair (HDR) to introduce a gene correction into the genome of cells using donor plasmid. They make a cut in the genome and the donor will correct the genome gene.
I am thinking of doing the opposite. I design a traffic light system in a plasmid to observe HDR in my cells. I put a transcription terminator in the homology region in the plasmid between the promoter and the GFP.
but there is no terminator in the homologous sequence in the genome.
CRISPR will cut the plasmid in a site which is homologous to the genome. By right, the HDR should use the genome to correct the plasmid gene, and the terminator should dissapear. I should observe the GFP turning on,
Do you think this system will work?
Will there be side effects like plasmid integration?
Interesting - so you’d have [promoter]-[homology with terminator sequence in it]-[GFP]? Maybe someone more knowledgable can chime in, but I can’t see why this couldn’t work theoretically if you don’t mind truncation of the target gene transcript. Might need to consider the CRISPR and HDR efficiencies (look at Scr7 and/or nocodazole to increase HDR if needed).
You say you’d cut the plasmid at a site homologous to the genome, but the disadvantage of that is that there’ll be two possible cut sites: one in the genome, and one in the plasmid. Meaning after HDR, your repaired plasmid can be cut again in the incorporated genomic sequence. Instead, I’d suggest slightly modifying the homology region to introduce a PAM (e.g. some synonymous mutation), and design your guide for that. This shouldn’t affect the HDR and will prevent Cas9 cutting the genomic sequence.
Also keep in mind that the plasmid will be degraded over the coming days post-transfection. In answer to your last question - random plasmid integration is always a possibility. Thermo puts it at ~1 cell per 10^4 transfected.
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