Title |
Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae |
Author |
Hye Yun Moon†, Gyu Hun Sim†, Hyeon Jin Kim, Keunpil Kim, and Hyun Ah Kang* |
Address |
Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 06974, Republic of Korea |
Bibliography |
Journal of Microbiology, 60(1),18-30, 2022,
|
DOI |
10.1007/s12275-022-1580-7
|
Key Words |
Saccharomyces cerevisiae, multiple integration,
marker-recycling, Cre-lox, CRISPR-Cas9 |
Abstract |
We evaluated the Cre-lox and CRISPR-Cas9 systems as markerrecycling
tools in Saccharomyces cerevisiae recombinants containing
multiple-integrated expression cassettes. As an initial
trial, we constructed rDNA-nontranscribed spacer- or Ty4-
based multiple integration vectors containing the URA3 marker
flanked by the loxP sequence. Integrants harboring multiple
copies of tHMG1 and NNV-CP expression cassettes were obtained
and subsequently transformed with the Cre plasmid.
However, the simultaneous pop-out of the expression cassettes
along with the URA3 marker hampered the use of Cre-lox as
a marker-recycling tool in multiple integrants. As an alternative,
we constructed a set of CRISPR-Cas9-gRNA vectors containing
gRNA targeted to auxotrophic marker genes. Transformation
of multiple integrants of tHMG1 and NNV-CP
cassettes by the Cas9-gRNA vector in the presence of the URA3
(stop) donor DNA fragments generated the Ura- transformants
retaining multiple copies of the expression cassettes.
CRISPR-Cas9-based inactivation led to the recycling of the
other markers, HIS3, LEU2, and TRP1, without loss of expression
cassettes in the recombinants containing multiple
copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively.
Reuse of the same selection marker in marker-inactivated
S. cerevisiae was validated by multiple integrations of the
TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1.
These results demonstrate that introducing stop codons into
selection marker genes using the CRISPR-Cas9 system with
donor DNA fragments is an efficient strategy for markerrecycling
in multiple integrants. In particular, the continual
reuse of auxotrophic markers would facilitate the construction
of a yeast cell factory containing multiple copies of expression
cassettes without antibiotic resistance genes. |