Simultaneous multiplex genome editing in yeast

A method for performing simultaneous multiplex RNA-directed nuclease editing in yeast cells comprising:

• designing and synthesizing ribozyme-containing editing cassettes that comprise regions of homology to a vector backbone for gap-repair insert of the ribozyme-containing editing cassettes into the vector backbone, wherein the ribozyme-containing editing cassettes comprise from 5′ to 3′:
• a pol II promoter;
• a transcription start site;
• a first editing cassette wherein the first editing cassette comprises a coding sequence for a first gRNA and a coding sequence for a first donor DNA, wherein the first donor DNA comprises a rational, desired edit to a first target sequence and an edit configured to render inactive a first proto-spacer motif (PAM) in the first target sequence;
• a linker;
• a second editing cassette wherein the second editing cassette comprises a coding sequence for a second gRNA and a coding sequence for a second donor DNA, wherein the second donor DNA comprises a rational, desired edit to a second target sequence and an edit configured to render inactive a second proto-spacer motif (PAM) in the second target sequence;
• a coding sequence for a self-cleaving ribozyme;
• and a pol II terminator;
• amplifying the synthesized ribozyme-containing editing cassettes;
• providing the vector backbone;
• transforming the yeast cells with the amplified ribozyme-containing editing cassettes and the vector backbone where gap-repair combines the amplified ribozyme-containing editing cassettes and the vector backbone to create editing vectors;
• and
• providing conditions for RNA-directed nuclease editing in the yeast cells by the editing vectors.