Cas9-SAM sgRNAs repressing gene expression

We cloned six sgRNAs targeting the human TET3 gene and transduced primary dermal fibroblasts which express low levels of TET3 (CT values ~35). We observed gene repression. Some of the guides gave ~2 to 4 fold gene activation in transient transfection of HEK293Ts (endogneous TET3 CTs are ~27). It seems that the TSS of TET3 is ill defined and the gene models predict multiple TSSs which can vary by 10 kb or more.

I am tempted to speculate that the repression is caused by the binding of the guides to the genebody instead of the sequence immediately upstream of the TSS.

Alternatively, is it possible that there is a post-transcriptional regulatory mechanism that controls the level of TET3 mRNA (a microRNA or LINC RNA which is expressed specifically in fibroblasts).

I would like to kindly ask for your thoughts on our results.


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Fuchsia Kelpiealmost 3 years ago

Could you please clarify whether you are transfecting sgRNAs with CRISPRa (SAM?) or CRISPRi (KRAB) in your experiment? It sounds like you are doing CRISPRa, but just want to make sure.

We have observed that dCas9 alone, when targeted downstream of the TSS, can produce a significant amount of repression (this is how I design my sgRNAs for repression). The TSS's for lncRNAs generally are not particularly well defined, and vary between cell types so they are tricky to activate. We have seen cases in which, even when the lncRNA TSS's are accurately annotated, that the

Mint Satyralmost 3 years ago

Thank you very much and I apologize about the delayed response. We are using CRISPRa (SAM). I will try your recommendation.

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