Help: can not detect Cas9 after infection with lentiCas9-Blast

I packed lentivirus from HEK293T cell line with lenticas9-blast #52962. I started selection with blasticidin 3 days post infection. Control group were dead and the infected cells were selected over one week. But it took at least one week to recover the cells (had to withdraw Blast) to have a decent number of cells to run WB ( is this normal?). However, when I did western blot to look at the expression using anti-FLAG (could detect both N- and C-terminus) and anti-Cas9 (ab191468, the one listed on the protocol). Only my positive control (protein of Cas9+ cells borrowed from neighbour lab) had the bands. I'm wondering why? For example, anything tricky for EFS promoter in leukemia cells or Blasticidin selection process? (I have sequenced/digested my prep of plasmid and am running transient transfection to check protein and all looks fine). Or is there any available Cas9 plasmid (better to be with colors, and does not have puro+ selection ) that could be used with the Gecko v2 crispr library (puro)? Thanks for any information ahead!!

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Sand Werewolfover 5 years ago

Try RT-PCR to see if you can detect Cas9 mRNA - be sure to run a no-RT reaction to control for possible amplification of integrated Cas9 DNA.

I have been using lenti dCAS-VP64_blast (addgene 61425) and haven't been able to detect Cas9 by western despite robust activation of my targeted gene. I have been able to detect Cas9 mRNA by RT-PCR.

You can also try testing Cas9 activity by lentivirally transducing with pXPR_011 (addgene 59702) which encodes GFP and a GFP-targeting sgRNA (as well as puro resistence). Reduced GFP expression means you have functional Cas9 expression.

Aquamarine Wyrmover 4 years ago

I have encountered a similar issue, where I cannot detect Cas9 in a B cell lymphoma cell line by western blot after transduction using lenticas9-blast. Since you posted this over a year a go I was wondering how did you resolve the issue in the end?

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