Hello,
I hope to insert a gene into mammalian cells in culture using CRISPR/Cas9 and an HDR template. Will insertion of a 1 kilobase gene cause chromosomal abnormalities and effect cell proliferation/viability? Is this one of the major concerns preventing the use of Cas9-mediated insertion as a therapy in humans?
Thank you.
Hi David,
The chance of chromosomal abnormalities is actually not the reason. The problem lies within the DNA repair mechanism itself. HDR frequency is very low in most of the primary cells and it is cell cycle specific (G2/S phase). The efficiency is better when you use ssODNS. But when you use plasmid to have a targeted insertion, the efficiency is too less. This is why most of the HDR based approaches fail to show their efficacy even though they are safe.
- Badges
- Science topic