Hello,

we want to mutate a gene in mammalian cell with CRISPR Cas9 and use Next Generation Sequencing (NSG) with Illumina to see the efficiency. We would also like to know the percentage of cells with mutations in both alleles (ko/ko), in one (ko/wt) and with none (wt/wt). We are not experts and we've been told that for this we'd need to do single cell-NSG, but I was thinking that if we can assume that the probability of mutation is the same for all the pooled alleles from all the cells, just by knowing from the NGS the % of mutated sequences (for example 25%=1/4), we could easily calculate the probabilities and thus the frequencies of the genotypes, couldn't we?

Probability of ko/ko=1/4 x 1/4=1/16 --------------->7,7% of the cells would be ko/ko

Probability of ko/wt=1/4 x 3/4=3/16 ---------------> 23% of the cells would be ko/wt

Probability of wt/wt=3/4 x 3/4=9/16 ---------------> 69.2% of the cells would be wt/wt

Sum=13/16 =100%

But maybe if one cell gets transfected by the sgRNA and the Cas9 this raises the probability that both alleles get mutated, and thus we cannot do that assumption, and the probability of ko/ko is actually higher? I imagine that there are many reports that by combining single cell sorting with validation methods have reported these probabilities, but I am not able to find such a basic thing among so much cutting edge litterature. If there is such a bias, is it more or less constant, and thus it can be used to calculate expected genotype frequencies, or it is very variable among different transfections/experiments?