I want to create a knock-down, stable cell line using crispr-cas system without using viral based vectors. I want to regulate the knock-down intensity around 60% so that I can do my experiments with the cell lines. How can I do it ??
You could for example make a GFP knockin in your locus using Crispr-Cas, transfect your cells, select them, pick single clones and determine the expression levels of your gene of interest.
Crispr doesn't knock things "down", but out... That said, it's pretty common that a hetarozygous knockout will have some percentage decreased expression, with some luck it might even be 60. You can transfect plasmids encoding the crispr + guide RNA directly, without the use of viruses. You'll be limited by your transfection efficiency.
More typically, if 60% knockout is your goal (and not 100%), then you'd consider yourself lucky that you can use interfering RNA, rather than having to resort to stable knockout cell line development.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line