Directed evolution: Point mutation vs Insertion-Deletion vs Shuffling?

Question

When attempting enzyme function improvement via Directed Evolution I can see three different strategies to generating variation for the gene sequence: 1. Point mutations 2. Insertion / Deletions 3. ShufflingIs there a systematic difference between how successful each of these approaches are likely to be for a particular gene or in general? Does one choose one over the other approaches in any specific project based on any characteristics / goal differences?A related question: On a methodological front, I know one can use error prone PCR to get #1 i.e. point mutations. What are similar techniques for #2 & #3? i.e. How does one execute gene shuffling or Insertion / Deletions in practice?

Answer 1

A pretty common technique is to just use rounds of radiation or error-prone PCR (like you mentioned) followed by some kind of stressor used to select for the effect the researchers want to see if something resistant to that stressor evolves. So the mutations will typically be random instead of designed. You could have point mutations, insertions/deletions, shuffling, etc. The researchers usually won't know what happened to the genome until they find a mutant that meets the criteria they want and sequence its DNA.