Aqua Imp
posted about 3 years ago

Should I maintain Antibiotics (Blasticidin and Puromycin) Selection throughout the screen?

I am currently working on a CRISPR screen using the Brunello Library, 2-vector system. The Cas9 portion contains Blasticidin selection and the sgRNA library has puromycin selection.

After infection and antibiotic selection (with both Blast and Puro), we did several rounds of FACS. However, after sorting, we found that those sorted cells gradually losing Blast and Puro resistances.

I wonder if I should keep both Blast and Puro selection throughout the screen, or it is okay to maintain the cells in regular media after the initial selection since the sgRNAs are already integrated and the genome has been modified.

Thanks a lot in advance!

Fuchsia Kelpieabout 3 years ago

We usually see the indels saturate at ~1 week after transducing the lentivirus, so after the initial selection of 1 week, the genome should have already been modified, so we do not typically keep the cells on selection during the screen. Keep in mind though, that you do not want to maintain these cells for too long (i.e., >2-3 weeks) because for genes in which the KO reduces cellular fitness, you may slowly see a reduction in indel rates for these genes in your cell population.

Aqua Impabout 3 years ago

Thank you so much!

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