Recently, I've done gene deletion in Komagataella phaffii using the CRISPR/Cas9 system with a Zeocin selection marker, but I've encountered an issue that I hope someone can help with.

Here’s the process I’ve been following:

Following this protocol, in my case, I consistently find that colonies grow on both YPD and YPD+Zeocin plates, suggesting that the Cas9 plasmid is not being eliminated, even though I confirmed gene deletion by gel electrophoresis. I've also tried increasing the number of growth cycles from 3 to 5, but the issue persists, and also transformed a fresh batch of donor DNA and Cas9 plasmid into new competent cells, but the problem still there.

I've reviewed several articles, but I haven't found direct information addressing this specific issue.

Does anyone have experience with CRISPR/Cas9 in K. phaffii or similar systems and can suggest a strategy to effectively remove the Cas9 plasmid at the final stage? Any advice or insights would be greatly appreciated.