We had a similar phenomenon in a CRISPR zebrafish line. The induced mutation made about 20 'nonsense' amino acids before the stop codon. We recently published our work, so this wasn't a problem. I suggest you examine three things:

First, where did the mutation occur? If it's close to the 5' end, most of the native protein will never be made. A 50 amino-acid chimeric end doesn't much matter if 500 native amino acids have been truncated off. In our case, over 90% of the proteins' native amino acids were eliminated.

Second, in silico BLAST the nonsense amino acids against all known proteins and see if there are any active domains or motifs. If there are none, this piece of protein probably has no effect at all. We found no matches and this is what we reported.

Finally, do a Western blot to show that your modified cells are not making any / are making less of the target protein. This assumes you have an antibody that recognizes the native protein very well.

Hi Elizabeth,

Thank you so much for your response. I feel more confident knowing that others have encountered this situation. Our particular mutation occurs quite early in the catalytic domain of the protein, so I would imagine that much, if not all, of the protein function would be eliminated. I did not think about doing an in silico BLAST analysis of the nonsense amino acids, thank you for the suggestion. Finally, I was planning on doing a western blot with 1-3 different antibodies that recognize a region of the protein preceding the CRISPR modification, before ruling out this mutant (as well as an RT-qPCR analysis). Thank you for your valuable input!

Best,

Es