I performed CRISPR/Cas9 in CHO cells to KO the protein Arrestin3. I transfected cells with a single plasmid containing gRNA - Cas9 - YFP. Then I sorted for YFP and I screened clones by sequencing. I got only 2 clones (out of 8) were there was a mutation (point mutation). I'm afraid that this will not be enough to get disruption of protein expression. Anybody know how could I improve the probability to get insertion or deletion?
Many thanks
My modest experience with CC9 tells me that in order to get a protein KO you would need to generate an insertion or deletion (in-del), to disrupt your Open Reading Frame (ideally close to the start ATG). Also, you need to KO both copies of the gene this way. If you can select for such clones, then you will have a KO line.
Try using two gRNAs at the same time targeting an important domain or region in your protein. Although this approach requires that the two cut events happen in the same DNA molecule, in my experience it actually occurs pretty often.
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