REFERENCES
Boisnard S., Zhou-Li Y., Arnaise S., Sequeira G., Raffoux X.,
Enache-Angoulvant A., Bolotin-Fukuhara M., Fairhead C. (2015). Efficient
Mating-Type Switching in C. glabrata Induces Cell Death. PLOS ONE
10 (10): e0140990
Cen, Y., Timmermans, B., Souffriau, B., Thevelein, J.M., and Van Dijck,
P. (2017). Comparison of genome engineering using the CRISPR-Cas9 system
in C. glabrata wild-type and lig4 strains. Fungal Genet. Biol.
107, 44–50.
Dorfman, BZ. (1969). The isolation of adenylosuccinate synthetase
mutants in yeast by selection for constitutive behavior in pigmented
strains. Genetics 61(2):377-89.
Dujon, B., Sherman, D., Fischer, G., Durrens, P., Casaregola, S.,
Lafontaine, I., De Montigny, J., Marck, C., Neuvéglise, C., Talla, E.,
et al. (2004). Genome evolution in yeasts. Nature 430, 35–44.
Enkler, L., Richer, D., Marchand, A.L., Ferrandon, D., and Jossinet, F.
(2016). Genome engineering in the yeast pathogen C. glabrata using the CRISPR-Cas9 system. Sci Rep 6, 35766.
Gabaldon T, Martin T, Marcet-Houben M, Durrens P, Bolotin-Fukuhara M,
Lespinet O, Arnaise S, Boisnard S, Aguileta G, Atanasova R, Bouchier C,
Couloux A, Creno S, Almeida Cruz J, Devillers H, Enache-Angoulvant A,
Guitard J, Jaouen L, Ma L, Marck C, Neuvéglise C, Pelletier E, Pinard A,
Poulain J, Recoquillay J, Westhof E, Wincker P, Dujon B, Hennequin C,
Fairhead C. (2013) Comparative genomics of emerging pathogens in theC. glabrata clade. BMC Genomics 14:623. doi:
10.1186/1471-2164-14-623
Gietz R.D., Schiestl R.H., Willems A.R., and Woods R.A. (1995). Studies
on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG
procedure. Yeast 11 , 355–360.
Knott, G.J., and Doudna, J.A. (2018). CRISPR-Cas guides the future of
genetic engineering. Science 361, 866–869.
Lemos, B.R., Kaplan, A.C., Bae, J.E., Ferrazzoli, A.E., Kuo, J., Anand,
R.P., Waterman, D.P., and Haber, J.E. (2018). CRISPR/Cas9 cleavages in
budding yeast reveal templated insertions and strand-specific
insertion/deletion profiles. Proc Natl Acad Sci U S A 115, E2040–E2047.
Maroc L, Fairhead C. (2019) A new inducible CRISPR-Cas9 system useful
for genome editing and study of double-strand break repair in C.
glabrata . Yeast. 36(12):723-731. doi: 10.1002/yea.3440.
Muller, H.; Dujon, B.; Fairhead, C. Comparative genomics in
hemiascomycetous yeasts. (2007). In “Candida: comparative and
functional genomics” (Eds: C. d’Enfert and B. Hube.), Caister Academic
Press, Poole, UK. pp.71-92.
Ralser M, Kuhl H, Ralser M, Werber M, Lehrach H, Breitenbach
M, Timmermann B. (2012) The Saccharomyces cerevisiae W303-K6001
cross-platform genome sequence: insights into ancestry and physiology of
a laboratory mutt. Open Biol 2(8):120093
Rodrigues, C.F., Silva, S., and Henriques, M. (2014). Candida
glabrata : a review of its features and resistance. Eur. J. Clin.
Microbiol. Infect. Dis. 33, 673–688.
Vyas, V.K., Bushkin, G.G., Bernstein, D.A., Getz, M.A., Sewastianik, M.,
Barrasa, M.I., Bartel, D.P., and Fink, G.R. (2018). New CRISPR
Mutagenesis Strategies Reveal Variation in Repair Mechanisms among
Fungi. MSphere 3.
Yarrington, R. M., Verma, S., Schwartz, S., Trautman, J. K., & Carroll,
D. (2018). Nucleosomes inhibit target cleavage by CRISPR-Cas9 in
vivo . Proceedings of the National Academy of Sciences of the United
States of America, 115, 9351–9358.
https://doi.org/10.1073/pnas.1810062115
Zordan, R.E., Ren, Y., Pan, S.-J., Rotondo, G., De Las Peñas, A.,
Iluore, J., and Cormack, B.P. (2013). Expression plasmids for use inC. glabrata . G3 (Bethesda) 3, 1675–1686.
Legends to figures:
Figure 1: Schematic representation of plasmid pCFYF. Elements shown not
to scale. The plasmid contains origins and selection markers forE. coli : ori and AmpR, as well as for yeast: ARS-CEN andURA3 . The Cas9 gene is under the control of the inducibleMET3 promoter. The gRNA gene can be cloned under the control of
the constitutive SNR52 promoter.
Figure 2: Survival on solid induction medium. Survival is calculated as
the ratio of the number of colonies that grow on plates with induction
medium (SC-Ind) over the the number of colonies that grow on plates with
repression medium (SC-Rep). Black bars: untransformed yeast (with Uracil
added to the media); dark gray bars: yeast transformed with the plasmid
expressing Cas9 but where no gRNA is cloned; light gray bars: yeast
transformed with the plasmid expressing both Cas9 and the gRNA. Error
bars represent standard deviation.
Figure 3: Survival to limited induction time and efficiency of
induction. Cells are placed in liquid SC-Ind medium for the time, in
hours, shown on the graph, followed by plating on repression medium
(YPD-Rep). The upper survival curve, starting at 100 %, is the ratio of
colonies on YPD-Rep at a given time-point over the number of colonies on
YPD-Rep at T0. This is normalized by division with the survival rate in
the same conditions, of cells transformed with pCFYF (expressing Cas9
but no gRNA). The lower curve starting at 0 % for C. glabrata and C. nivariensis , and at 40 % for C. bracarensis is the
percentage of red colonies on total colonies on SC-Rep medium. Error
bars represent standard deviation.
Figure 4: Sequence of NHEJ junction in Ade- cells of the three species.
First line shows sequence before the DSB in all three species, with
double arrow indicating cut site, and letters in italics, the PAM
sequence. Coordinates relative to the first base of coding gene is
shown. Following lines show sequences after unfaithful NHEJ events, with
letters in bold indicating insertions and dashes indicating deletions,
the (-1) indicating deletion of the nt at position 471. The frequency of
each repair event is indicated on the right.