If the CUG is upstream of the intended Met start, then you don't need to worry what to change it to, since it is not being translated into a protein. You can either delete a base or change either the U or G to anything else. If you have internal CUG codons then you can change those to any other Leu codon and you won't change your protein. If you have internal AUG starts then you need to judge from the context of the protein what amino acid to change it to, but usually internal shorter restarts do not result in active protein so they might not be a problem. 

Hi Michael,

Thank you for your reply.

In my case, the CUG is upstream of the Met (AUG) start. So I want to have cells that only express one isoform: AUG or CUG.

For the first scenario (AUG only), I was planning on doing as you suggested "change either the U or G to anything else" of the CUG codon.

For the second scenario (CUG only), I am still not sure whether Leu would be the most suitable conversion for the downstream Met (AUG).

In addition can you please advise whether the CRISPR-Cas9 system would be the most suited technique or should I consider something else.

Thank you!

It is difficult to say which is the most suitable amino acid to replace Met, because it very much depends upon what role the Met has in the protein. On the basis of hydrophobicity then Leu is as good a guess as anything. But if the sulfur is playing a role in catalysis then it might not be. So without more information it is just a guess. 

Whether CRISPR-Cas9 is the best system also depends upon your experimental setup. Is this something cloned in a plasmid, in which case traditional site directed might be easier. If this is a chromosomal locus then CRISPR might be good, but again is this E. coli or is this a less manipulable organism. 

Hi Michael,

I intend to mutate the AUG codon in mammalian cells (probably will start with HeLa cells). I'm hoping to then have cells lines that only produce one isoform, i.e. the CUG-translated isoform only.

In the CUG translated isoform, I am not sure what role the internal Met is playing. I do have an expression plasmid where we have converted the internal Met to Leu, but have not put it through any functional assays. I do plan on doing that next.

However is Leu the best replacement for Met, or are there any others?

Thanks,

Jerry