Hello everyone,

I used IDT Alt-R system to make my RNP complex with gRNAs designed from CRISPOR tool. I showed guides are working with 100% efficiency by in vitro cut efficiency test. After I performed my IUE to mouse brain embryos, I dissociated cells from the tissue and FAC sorted them according to the co-transfected plasmid signal. Then I performed fluorescent PCR with capillary gel electrophoresis analysis to evaluate Cas9 cut efficiency. I did obtained 6% cut resulting in out-of-frame, however I also performed IHC to the electroporated brain slices and observed a very strong phenotype compared to the LacZ control. 6% cut seems incredibly low to me to observe such a strong phenotype change. I could't find a way to explain the reason for this result.

I attached the papers I followed for fluorescent PCR procedure and using the same conditions for IUE in the other paper.

Do you have any idea what could be the reason? Or have you also experienced such a thing before?

Many thanks in advance.