Hi everyone,

I'm trying to obtain a heterozygous clone (wt/KO) using a CRISPR-Cas9GFP RNP approach on iPSC-Like. After the nucleofection, 90% of cells are GFP+. After sorting cells, only 4% have a wt allele. I did a clone selection to find a wt/KO but none have this genotype. Worse still, no clone has at least one wt allele. (I analyzed a lot of clone). Do I have to use a gRNA with a lower ON-Target score ? I cannot use a plasmid because the lipofection efficiency on these cells is 3%. Thank you.