Puromycin selection for transient transfection of sgRNA

Lv Wei

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Nov 13, 2015, 10:49:56 AM11/13/15

to Genome Engineering using CRISPR/Cas Systems

Dear all,

If I did the transient transfection of sgRNA plasmids into cells, for example using PX459, how long should the puromycin selection last? Because it is not a stable expression of puromycin, for long time cultivation from single cell to cell cluster, should I add puromycin in this period? I was a little confused.

Thank you to you all.

Best,

Lu

YIng Dang

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Nov 13, 2015, 12:32:37 PM11/13/15

to Genome Engineering using CRISPR/Cas Systems

Puromycin works very well with transient transfection. A small portion of cells will always keep their puro-resistance no matter how long you culture them. And those cell are your enriched constantly sgRNA-expressing cell. If you have co-transfected cas9 plasmid, you will end up getting extremely high Knockout efficiency. Well, off-target effect will be another issue.

Good luck,

Ying

Lv Wei

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Nov 13, 2015, 1:39:03 PM11/13/15

to Genome Engineering using CRISPR/Cas Systems

So, you recommend keep using puro+ medium in culturing the single cells. OK, thank you so much.

在 2015年11月13日星期五 UTC-5下午12:32:37，YIng Dang写道：

Rocamar

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Nov 13, 2015, 2:23:57 PM11/13/15

to Lv Wei, Genome Engineering using CRISPR/Cas Systems

I prefer to remove puro when my ctl , non transfected cells are dead...and it works . Keeping the puro will select cells with integrated puro plasmid ..this is not so nice .

So I advice to remove the puro.

Didier

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YIng Dang

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Nov 13, 2015, 3:49:57 PM11/13/15

to Genome Engineering using CRISPR/Cas Systems, greenyg...@gmail.com

Cells survived in puro selection are cells that kept puro-cassette plasmid. However, when them expanding, there is only one cell can keep that plasmid. Cells survived AND expanded are cells that have puro-cassette integrated in their genome, while this happen in very low frequency. So all depends on your own design.