Dear researchers,
I booked two crispr/cas9 plasmids to knock out a specific gene, however, after selection, the western results showed me that, one is not working, another one there are still some protein left ? Does anyone know why there is still protein there？ Does it mean that crispr/cas9 didn't work?
If there is still protein left, this means that it didn't work out or that your knockout is only heterozygous. Did you use T7E1 assay to check for cleavage?
Dear Ariane, thank you for the the answering, I didn't do the T7E1 assay to check for cleavage, I will google the protocol and do it as quickly as possible.
I would recommend to do this! It's easy and fast und you will get to know if your CRISPR is working or not! If it's working then it seems that you just isolated some cells where the knockout was only heterozygous.
Thank you again, one more question, do you think I can use the cells for further experiment, if the knockout was only heterozygous?
@ Bin Liu, thank you for answering me. I use antibiotic selection. And I did the T7E1 assay, the result shows me that there is cleavage. Since knockdown is enough for me to study the gene function, I just wondering in the future some editors will ask why Crispr/Cas9 didn't knock out the gene only knock down.
I am having a similar problem after FACS sorting of the plasmid gRNA/Cas9 with GFP, I wanted to check if there is knockout since GFP positive shows that only plasmid transfection works, so I am doing single cell cloning but I have more than 100 samples and using WB, sequencing or T7E1 assay will cost me a lot, I would be grateful if I anyone could suggest me a cheaper and efficient way.
Until now I also always had to screen a lot of clones, but I don't know another way to do. I'm using WB at first, because it's way cheaper than the T7 Assay and you can directly see if your protein is there or not. In the T7 Assay you cannot always discriminate between heterozygous and homozygous knockout.
One important subject to know when you are handling CRISPR/Cas9 experiments is the ploidy of your cell lines. Specially, if you are working with cancer cell lines, they might bear chromosomal aberrations like hypertetraploidy. This feature make it harder for Cas9 generate a true knockout trough DSB.
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