Dear expertise

I am trying to clone one metabolic pathway (4 genes). Two cassettes carrying homologous sequences (50 bp) on 3 and 5 prime terminus to flanking each other and the other ends homologous to up and downstream of a double strand break by CAS 9 into a target gene on genomic DNA, in order to knock out this gene during recombination, of industrial diploid Saccharomyces cerevisiae using multiplex CRSPER .

I succeeded to transport both pCAS plasmid - KanMX (carrying target sgRNA, and Cas 9) into S. cerevisiae...where the colonies succeeded to grow on G418 and also I confirmed a transport of the linear repairing DNA (cassettes) by PCR... Within one or two generations of these positive colonies on YPD liquid medium, I found the selected colonies lost their linear cassettes..... Do you have explanation for this case???

I just guess both pCAS plasmid and linear DNA was transported during transformation, but the linear DNA wasn’t able to integrate into genomic DNA and just transport inside cells after transformation and then lost.

Does my guess is correct? Or something else was occurred...e.g. DNA repairing system is faster than homologous recombination in these diploid cells. What should I do?

Thanks in advance for your precious reply

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