It depends on what the TF recognises and binds. If you are not sure, you have to test this systematically.

Thank you Angela , so my thoughts are as fellow :

For example , my TF recognises TGATGXAAX, i want to make the middle G into A to break this pattern . do you think it could happen that one is not enough that i need to make more ? or i make two mutation at the first time .

I am not sure if there are any rules about this kind is experiments if not i guess try more than one mutation will be better chance to get result .

thank you !

Do you have any information on what your TF looks like? DNA-binding proteins usually have canonical folds which allow them to interact specifically with DNA e.g zinc fingers. If you want to see how it binds you could always make a model from a homologue whose structure was solved with DNA- bound. This may be more informative for deciding which DNA bases are important. Otherwise you may have to make a series of mutations and then do your test e.g middle G altered, TG altered, etc to narrow down the specific base-protein interactions.