I am trying to KO a gene in liver as well in cell line, using 3 sgRNA cloned into an all-in-one lentivirus with cas9 in it, but somehow I did not see any expression change, any suggestion?
Hi Peter,
Couple questions.
1) Are you trying to knockout a gene in primary hepatocytes and another cell line? One possibility is that this gene is essential for the survival of these cells, and therefore you cannot achieve a successful CRISPRR knockout. Is there literature demonstrating the essential nature of this gene in your target cells?
2) Did you align your guide sequences against the reference genome to make sure they are targeting your gene of interest? You can either use Ensembl or NCBI Nucleotide BLAST for this.
3) Are you checking for expression change via Western blot in single-cell colonies? After transfecting my target cells with the CRISPR vector, I got them single-cell sorted into 96-well plates and then screened these single cell colonies for knockout. About 10% of all colonies exhibited decreased protein expression compared to control cells- so knockout efficiency wasn't that high.
I have a well working protocol for CRISPR KO, so feel free to talk to me more for troubleshooting.
Best,
Nesli
Deniz Nesli Dolcen Thank you very much
1) it is ADH gene which should be not essential at all. people have done that in cell line. 2) yes, gRNA is designed with CHOPCHOP and they are correct. 3) I am using western blot to check expression. I can't use single colony since my purpose is to select the the gRNA sequence with the highest efficiency, and fir this reason I can't use any selection marker either.
how many gRAN sequence do you normally use in your KO experiment? I understand some gRNA are good, some are less effective on KO
Number of cells with ADH knockout in a heterogeneous (not single cell-sorted) cell population would be too low to yield a noticeable decrease in protein quantity on a Western blot. Speaking from experience and from what I have been told by others who have done CRISPR KO, you can expect 10-20% of cells in a transfected cell population to exhibit either a mono-allelic or bi-allelic knockout of your gene of interest. Best case scenario: if all of these knockouts are bi-allelic, meaning 10-20% of cells lose the gene/protein completely, then the most you could observe is a 10-20% decrease in protein quantity on a Western blot. However, in reality, some of these cells will be mono-allelic knockouts.
If you want to assess gRNA efficiency without doing single cell sorting, you will have to perform NGS DNA sequencing on your heterogeneous cell population to detect low level indel events. That will be pretty expensive however.
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