Does it make a difference if you knock enemies out instead of killing them?

Solution 1:

The only difference between killing and knocking the opponent out is that, knocking them out can give you a moral boost to the ending of the game when you are trying to get the Redemption(Do the correct moral things to fix what you done in the past.)-100G achievement or if you want to get the Shadow Ranger-70G achievement(Don't kill a single human in the game-Except the Flamethrowers in the last mission)

Solution 2:

First of all, it's higher chance to get good ending if you get through the game without killing creatures (even mutants, when you can avoid them) unless forced to.

There are some levels when you could kill humans freely:

In the train from Ganza, when you're with Khan, on the Red Square level and in the end, in D6

If you want to get rare achievement, which mentioned in comment, you shouldn't kill anyone at all, except on levels from the spoiler (I'm not sure, it's not tested by me yet). Also, you won't receive it even if you shot someone and then reload level, so you will need to start the game from the beginning.