Hi Goodwin,

I never had problems with px459 (SpCas9 T2A Puro and sgRNA) in HEK239T experiments. I usually apply puromycin selection transiently, from 24 to 72 hours post-transfection. This is sufficient to transiently select cells which were transfected successfully.

You don't normally need prolonged puromycin selection to avoid stable Cas9 integration since most editing events happen between 24 and 72 hrs post-transfection.

The fact that you cannot detect editing events might mean that your sgRNA is not very efficienct (or not expressed properly). Make sure you sequence your plasmid before transfection, and that your sgRNA cassette (right after the U6 promoter) starts with a G nucleotide (A might work too but G is usually required for transcription start from a U6).

Since you cannot detect the plasmid in the surviving cells, this imply the concentration of Puromycin you are using is too low, and some cells can escape selection, adapt and live happily without your plasmid being actually there.

Last thing is that, given the high levels of transfection in HEK239T, selection is normally not even necessary. The fact that you are not detecting your plasmid by PCR might also imply your transfection didn't work.

This is all I can think about

Hope it helps

Francesco

you may want try 24 h after transfection.

Thank you. I will try short duration in parallel to high dose puromycin. I am using Dr. Zhang Feng (Broad Institute) plasmid, so I guess cassette might be ok. I have sequenced the cloned oligos.