I have transfected mouse ES cells with single gRNA, Cas9 system. I am able to detect cleaved bands after T7 endonuclease. However, how to determine if this is monoallelic or biallelic mutation?
You will not be able determine this, since at this stage you are still working with a mixed population of cells. Some of the cells are edited at one allele, some at both, and some cells are not edited at all. You need to isolate single cell clones and then sequence a region of interest.
hI,
try CEL or surveyor mismatch cleavage assay
https://www.ncbi.nlm.nih.gov/pubmed/28876032
https://www.ncbi.nlm.nih.gov/pubmed/24445736
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349094/
https://www.ncbi.nlm.nih.gov/pubmed/25566793
.
Dear Evgueni, I have plated single cells and picked single colonies for genotyping
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