Hi,
I am trying to generate a homozygous knockout line for a particular gene in mouse embryonic stem cells by CRISPR/Cas9 mediated recombination. My strategy is to replace my target gene with GFP and then screen single cells post GFP sorting. Till now I have screened close to 100 clones but I haven't obtained even a single homozygous knockout. Since heterozygous clones are being formed, I am assuming that the gRNA designs are fine. What can I change in my stratgey/approach to maximize chances of obtaining a homozygous knockout? Also, how can I be sure that homozygous knockouts are being generated but they are just not surviving till the clonal selection process?