I want to generate a KO of my favourite gene using CRISPR-Cas9 in iPS cells. I already obtained an heterozygous clone but I was wondering if I could transfect this clone again to improve chances to have an homozygous one? Is this relevant?
Have you not obtained any homozygous clone with the first transfection? From my understanding of CRISPR/Cas9, if you are using HDR and your guide RNA is very specific then I believe that, indeed, it would be possible to obtain an homozygous clone by re-transfecting the heterozygous one.
Thank you for your answer.
I didn't have any homozygous clone, just one heterozygous. I used double nickase Cas9 with 2 guide RNAs which induces two single stand breaks that will be repaired by NHEJ and lead to indels. I think there is no reason not to try to re-transfect it...
You may need to do single cell cloning if you want to get homozygous. The chance of knocking out both allele is much smaller than knocking out one.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line