CRISPR SAM activation

Weijun Liu

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Aug 8, 2019, 1:18:21 PM8/8/19

to Genome Engineering using CRISPR/Cas Systems

We selected 3 gRNAs from http://sam.genome-engineering.org/database/ for each of our interested genes, I basically did all experiments as described in Konermann's 2015 Nature paper. Then we did qRT-PCR to test the gene expression fold change. However,half of the gRNAs (8/15) showed only 1~2 fold increase, only one showed 2.2 fold increase. Others showed less than one fold increase. I have to point out that the basal level of these genes in our tested cells is intermediately abundant. I want to know is my result as expected or is the SAM system in our hands failed? I am wondering what is the range of fold change by SAM activation for genes not weakly expressed? We though that SAM should induce at least 4 or 5 fold increase for most of the intermediately abundant expressed genes.

Thanks,

Weijun

Julia Joung

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Aug 14, 2019, 6:08:55 PM8/14/19

to Weijun Liu, Genome Engineering using CRISPR/Cas Systems

Hi Weijun,

When you say intermediately abundant, what is the expression level relative to a housekeeping gene like ACTB or GAPDH? The fold change that you are seeing for activation sounds very low, and I would expect at least 5 fold change as you suggested. Perhaps the transfection efficiency is very low for your cell type?

Best,

Julia

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Harkirat Singh Sandhu

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Jun 14, 2021, 6:29:53 PM6/14/21

to Genome Engineering using CRISPR/Cas Systems

Hi Julia,

I am facing a similar problem where the endogenous expression of my GOI is intermediate but there is NO upregulation with SAM. I have used individual as well as pooled gRNA from the Broad institute database, and followed the exact protocol. My helper vectors were GFP tagged and the stable cells looked pretty good after sorting for GFP+ cells. Could it be that all the 3 SAM components did not get expressed in all cells? Could it be solved by screening for sc clones? Now I am also trying teh antibiotic-resistant helper vectors to see if I am able to select for the +ve cells. Can you please suggest what could go wrong?