lentiGuide_Puro_sgGFP_plasmid

My lab recently ordered Human CRISPR Knockout Pooled Library (2 vector system) from Addgene and over the last few months, we have been preparing for our screen. We generated stably expressing Cas9 cells of our cellular model (Hek293) and even though the Joung et. al. protocol states that it is not necessary to generate clonal lines we decided to do so. WB analysis revealed fewer amounts of Cas9 in our positive clonal line vs. selected population of cells after transduction which raised some questions. Because of this issue, we are planning on performing a Cas9 activity assay on our clonal line using sgGFP since our model also stably expresses a GFP fusion protein. We plan to determine if our positive clonal line shows adequate Cas9 activity in order to proceed to the screen itself. For this purpose, we require a lentiGuide_Puro_sgGFP plasmid which unfortunately we can not construct since the library we ordered does not include an empty backbone of lentiGuide_Puro plasmid.

I'm wondering if anyone has an explanation for our WB results because according to our knowledge we expected at least the same if not the higher amounts of Cas9 in our positive clonal line as opposed to the selected population of cells (we transduced the cells with MOI=0,6)? Also, did anyone maybe construct the already mentioned lentiGuide_Puro_sgGFP plasmid because unfortunately, we cannot find it in the Addgene repository?


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Fuchsia Kelpieover 3 years ago

Could you please share how you constructed the Cas9 positive clonal line? That might help us narrow down why you are getting those results.

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