Two publications from March in Nature Biotechnology show that Scr7 can be used to favour homology-directed repair (HDR) over non-homologous end-joining (NHEJ) for CRISPR/Cas9 applications.
I've been trying to repeat this in K562 and HEK293T cells (one of the studies used HEK293 cells), but so far haven't seen any effects. I tried 0.01-10mM Scr7, adding the drug immediately after, or 6h/12h after electroporation and then replenishing the drug after 24h and 48h. But none of the conditions I've tried so far have had any effect compared to my control.
I was wondering if anyone might have experienced troubles with Scr7 stimulation as well?
Could a standard media compound (DMEM,10%FBS,10mM HEPES, 1% Pen/Strep) affect the drug activity? (I'm about to try antibiotic free medium next, but my hopes aren't high on that one)
The publications used Nucleofection or Fugene for their plasmid transfection. I am working with classic electroporation as we don't have access to a nucleofector and found that Fugene is not suitable for the delivery of single stranded HDR templates.
My electroporation buffer is high in potassium salts, and is diluted 1:20 in culture media after electroporation. Could salts from the buffer or cell debris from the electroporation step interfere with my drug's activity?
I would be very grateful for your input!
Cheers,
Astrid
Not being en expert on this, but it seems is not easily reproduced. See some notes for example here: https://groups.google.com/forum/#!forum/crispr . If something can affect the drug cinc. in the media, the FBS will be my first suspect. May be trying a FSB minus condition after EP is worth checking.
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