Is it possible for the next ATG in frame after the deletion of the ORF ATG still translates to a functional protein?
I'm not a geneticist but assuming there is still a functional promoter and RBS, I believe the downstream gene should be fine. However, for genes that follow a single promoter this may not be the case. An internal target by Cas9 results in dsDNA cut that gets repaired by host mechanisms usually causing base deletions or substitutions that make the gene now non-functional. Assuming these cuts and repairs don't affect the genes start and termination sites then a transcript and potentially a non-functional protein could be made. In this case, downstream genes shouldn't be affected. However, if the termination site of the upstream gene is lost this could result in read through transcription and cause a single transcript for multiple genes which could be problematic.
Mutations induced in the beginning of genes have the potential to translate into functional proteins. However to get null mutants, better to mutate the genes across their functional domains or perhaps in the middle of the gene. Such deletions result in significant protein length polymorphisms which lead to truncated proteins in the mutants with no function.
Special thanks for your valuable answer. I actually had an insertion as well and after translation had stop codons. Basically the mutant had a deletion of about 528bp including the first ATG, however after the 166th amino acid had another ATG which is in frame. Both mutant strains had phenotype difference from the WT as well as the expression of some reported genes were downregulated in the mutant strains
It is less likely that such big deletion (166 aa) still result in the same protein activity. Why not perform a western blot among the mutants and wild types to prove the protein and to rule out any isoforms. Also, BLAST the amino acid sequence against rundundant plant proteins using ncbi databse to explore if protein mutations lie in the conserved functional domains of your candidate gene, if yes, you have much more chances to get null mutants. Good luck!
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