Crispr activation system ( 2 vector system) did not upregulate the expression of gene of interest.

[ANISH CHAUHAN]

Hii everyone, 

I am doing masters from AIIMS new Delhi. Currently, i have done FAT1 gene overexpression using crispr activation (2 vector system) but i found  downregulation of gene expression instead of overexpression.

what should i do ?

[Hamish McWilliam]

Hi Anish,

Have you tried a few sgRNA? Not all sgRNA will work, so I usually try 3+ top ranking sgRNA that are placed at different sites upstream of the gene.I’ve also seen one sgRNA that decreased expression of a particular gene, it was close to the start codon and maybe this was a factor.

Cheers

Hamish 

On 14 Oct 2023, at 10:17 am, ANISH CHAUHAN <anishde...@gmail.com> wrote:

Hii everyone, 

I am doing masters from AIIMS new Delhi. Currently, i have done FAT1 gene overexpression using crispr activation (2 vector system) but i found  downregulation of gene expression instead of overexpression.

what should i do ?

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[ANISH CHAUHAN]

Not yet, i have done my experiments by using one of the three commercially synthesized sgRNAs. 

Thank you for your response.

[Karim Daliri]

Hi Anish

From which company you order gRNA?

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[Ryan Weiss]

Hi Anish,

Wanted to jump in here and mention that we have also had issues with the 2-vector system (Sanson et al). Interestingly, genome-wide screening with this system seemed to work well, but individual sgRNA validations gave varied activation results and most sgRNAs we tested did not seem to activate gene expression much above nontargeting sgRNA control. I would recommend switching to the 3-vector SAM system (Zheng lab). In our hands, this system is very robust and we have found upregulation of the majority of sgRNAs tested across different gene targets (same sgRNAs tested in the 2-vector system). We have not compared to the newer 2-vector SAM system yet.

Sincerely,

Ryan

[ANISH CHAUHAN]

I have used 2 vector system for overexpression of gene of interest. But I'm lagging my research work bcz I found empty lentiSAM plasmid downregulates the gene expression.

I had searched but i didn't get how to troubleshoot.

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[ANISH CHAUHAN]

Kindly suggest me  any online tool used to design efficient sgRNA negative control Sequences.