Hello genome editors!
I wanted to know what techniques you use to edit multiple gene loci at the same time. I want to do this in ES cells and target up to 5-6 loci at the same time. Is there an efficient way to do this?
Alternatively, is there a 2-step method that I can use? Detailed protocols would be very helpful to read through.
Thanks
Navneet
Sounds like not an easy job. I would suggest target 2~3 loci at a time, screen for positive colonies and then do the second round of transfection and screening. The chance to get all 5 gene knockout in single transfection will be quite low. Not to mention that you might need to try a few different guides in each target.
Exactly what I was thinking. However from what I've read, efficiency of CRISPR for changing the genetic code is around 1-10 %. For multiple loci, this will fall to very low levels and I will need to screen 1000s of colonies to find a positive clone.
Making deletions however might be easier I feel.
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