- Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae
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Hye Yun Moon† , Gyu Hun Sim† , Hyeon Jin Kim , Keunpil Kim , Hyun Ah Kang
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J. Microbiol. 2022;60(1):18-30. Published online December 29, 2021
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DOI: https://doi.org/10.1007/s12275-022-1580-7
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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.
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Citations
Citations to this article as recorded by  - A comparative review of vector insertion techniques in Saccharomyces cerevisiae
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- The nature of meiotic chromosome dynamics and recombination in budding yeast
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Soogil Hong , Jeong Hwan Joo , Hyeseon Yun , Keunpil Kim
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J. Microbiol. 2019;57(4):221-231. Published online January 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-8541-9
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1,004
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Web of Science
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Abstract
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During meiosis, crossing over allows for the exchange of genes
between homologous chromosomes, enabling their segregation
and leading to genetic variation in the resulting gametes.
Spo11, a topoisomerase-like protein expressed in eukaryotes,
and diverse accessory factors induce programmed doublestrand
breaks (DSBs) to initiate meiotic recombination during
the early phase of meiosis after DNA replication. DSBs
are further repaired via meiosis-specific homologous recombination.
Studies on budding yeast have provided insights
into meiosis and genetic recombination and have improved
our understanding of higher eukaryotic systems. Cohesin, a
chromosome-associated multiprotein complex, mediates sister
chromatid cohesion (SCC), and is conserved from yeast
to humans. Diverse cohesin subunits in budding yeast have
been identified in DNA metabolic pathways, such as DNA
replication, chromosome segregation, recombination, DNA
repair, and gene regulation. During cell cycle, SCC is established
by multiple cohesin subunits, which physically bind
sister chromatids together and modulate proteins that involve
in the capturing and separation of sister chromatids. Cohesin
components include at least four core subunits that establish
and maintain SCC: two structural maintenance chromosome
subunits (Smc1 and Smc3), an α-kleisin subunit (Mcd1/Scc1
during mitosis and Rec8 during meiosis), and Scc3/Irr1 (SA1
and SA2). In addition, the cohesin-associated factors Pds5
and Rad61 regulate structural modifications and cell cyclespecific
dynamics of chromatin to ensure accurate chromosome
segregation. In this review, we discuss SCC and the
recombination pathway, as well as the relationship between
the two processes in budding yeast, and we suggest a possible
conserved mechanism for meiotic chromosome dynamics
from yeast to humans.
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