gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR/Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events and diverse approaches for multiplex genome editing have been described in literature by means of Cas9 or Cas12a endonucleases and their associated gRNAs. The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native PolII or PolIII RNA polymerases. These gRNA-expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in S. cerevisiae. Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single DNA editing. These DNA fragments can be custom-ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR-toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other, less genetically accessible yeast species.