I am using CRISPR on human iPS cells to delete my gene of interest by using two guides that cut respectively in 5' and in 3' of the gene. I transfect with myrrus the two guides and the Cas9 and select with puro for two days. For now I managed to get only one heterozygote clone out of 50 and I will try to re-crispr it. This method is definitely not efficient for human iPS and ES cells at all as we also use it on mouse ES cells in the lab and it works really nicely (10% homozygote clones). Any tips or published efficient protocols for these type of cells?
With CRISPR/Cas9, after the puro-selection, I did limited dilution where I tried to grow one cell/well (96 well culture plate). After 3 - 5 days after limited dilution I screened my plates in order to identify the cells clone that was grown from a single cell growth. Out of 150 single cell clone I got 12 clones with mono-allelic genome editing. I increased the number of the screening and luckily I got one clone with di-allelic genome editing.
I did re-crispr but it did not work and there are a high possibility of off-target genome editing which may affect other cell biological functions.
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