I'm looking for explanations why I couldn't achieve a full KO of a gene.

I'm working on the KO of 4 genes in Nicotiana tabacum using CRISPR/Cas9. (It's a 4n allotetraploid, meaning 1 gene= 2 different variants of the gene = 4 alleles). There are two kinds of genes we tried to KO simultaneously, one of them is a group of 3 genes and the second group is another other gene.

While managing to fully KO the first group (3 genesX4 alleles), we didn't manage to create a full KO of the sole gene.

We have created so far 3 different constructs, trying 8 different gRNAs against this particular gene.

Important to note: Cas9 is active in the plants we have created and the plant can live without the gene (other publications in different models managed to fully KO this gene).

I'm wondering if it's due to methylation of the genome at the placed where the gRNAs attach/ happen to be 8 unsuccessful gRNAs?/ maybe there's another gene in the group that we have missed...?

Any ideas/suggestion what could it be?

Thanks!