Hi All, I am new at CRISPR/Cas9 field, I tried my first CRISPR KO cell line but it failed. I transfected CRISPR/Cas9-gRNA into HEK293T cells via lipofectamine 3000, after 48 hours, I extracted genomic DNA, amplified the target region and performed SURVEYOR assay, however, I can't see any cleavage. I presume either the transfection efficiency is low or the targetting efficiency is low. I used WT CRISPR/Cas9. Is there any good suggestion how it happens and what I should do to improve it? many thanks!
Hello Wang,
my lab also use CRISPR system. to determine whether our KO has worked we wait for at least 5 days and then stain endogenously for our protein of interest.. we test several CRISPR constructs that targets our gene of interest. once we have proved this by IF we then transfect cells with the appropriate constructs and then sort for single cell population. once cells grow we perform IF again to determine which of the clonal cells indeed have a KO. based on this we move to perform genomic DNA expt,
with my experience so far, i think the 48 h might not be enough for complete KO. Also, there would be a mixture of the cells that are truly KO even in the transfected cells and some will not. So, i suggest longer period of incubation for the crispr construct and possibly flow cytometry sorting might help.
also Horizon genomic do offer free crispr plasmids which you can test to identify which of them works but you will have to license the clonal cell line to them should it work.
bw
Hi Valentina, thank you for your suggestions. Yes, I now switched to another CRISPR/Cas9 vector expressing puromycin marker, I will select transfected cells with puromycin first before single colony culture. Actually I am always worried about the necessity of the expression of my gene of interest, especially when I need to generate a homozygous cell line, absence of two copies might be lethal to cells......
Hi all,
I am also working with a Double Nickase Plasmid of CRISPR/Cas9. Unfortunately, I didn't get a good efficiency of transfected cells knocking out so in order to confirm wether the problem is my cell line i am going to try the same transfection in HEK293 cell line. After the selection with puromycin, does anybody know if is there any other way of checking transfection appart from a WB analysis with anti-Cas9? I will first try with the control plasmid.
Thank you very much in advance!
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