Publications by authors named "Nicole Xanthe Bennis"

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gEL DNA: A Cloning- and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing.

CRISPR J 2021 Apr 23. Epub 2021 Apr 23.

Department of Biotechnology, Delft University of Biotechnology, Delft, Netherlands.

Even for the genetically accessible yeast , 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 the literature by means of Cas9 or Cas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III 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 . Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex 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 and promises to be relevant for other less genetically accessible yeast species.
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April 2021