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Using CRISPR/Cas platform for Genetic Modification of Commercial Saccharomyces cerevisiae strains
C. Eicher, Géraldine L. Klein, Pascale Winckler, Jean-Luc Parrou, Hervé Alexandre, Cosette Grandvalet
To cite this version:
C. Eicher, Géraldine L. Klein, Pascale Winckler, Jean-Luc Parrou, Hervé Alexandre, et al.. Us- ing CRISPR/Cas platform for Genetic Modification of Commercial Saccharomyces cerevisiae strains.
OENO-IVAS 2019, Jun 2019, Bordeaux, France. �hal-02466806�
Using CRISPR/Cas platform for Genetic Modification of Commercial Saccharomyces cerevisiae strains
Eicher C. 1 , Klein G.L. 1 , Winckler P. 1 , Parrou JL. 2 , Alexandre H. 1 , Grandvalet C. 1,3
1 UMR A 02.102 Procédés Alimentaires et Microbiologiques, AgroSup Dijon – Université de Bourgogne Franche-Comté, Dijon, FRANCE
2 INRA - CNRS - INSA Toulouse, UMR 0792 LISBP Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés. Centre de recherche de Toulouse, Toulouse, FRANCE.
3 Institut National Supérieur des Sciences Agronomiques, de L’Alimentation et de L’Environnement, AgroSup Dijon, Dijon, FRANCE
Background: Traditional wine fermentation is a complex microbial process initiated by various yeast species classified as Saccharomyces and non-Saccharomyces species. To better understand the different interactions occurring within wine fermentations and track a specific yeast population, we wish to obtain GFP tagged yeast cells that stably express fluorescence signal without compromising the fermentative capability of the strain. To this end, the CRISPR/Cas9 system was investigated to genetically modify the commercial Saccharomyces cerevisiae diploid strain Lalvin EC1118® (LALLEMAND OENOLOGY).
The emergence of the CRISPR/Cas9 system has revolutionized genome-editing technologies.
Recently a set of vectors for simultaneous expression of Cas9 with gRNA cassette enabling simple and fast deletions of genes have been developed (Generoso et al., 2016). To modify genetically commercial yeast, the CRISPR/Cas9 machinery, along with a homologous donor sequence, were undertaken to insert eGFP gene into the EC1118® AMN1 gene. The AMN1 gene, which encodes a protein required for daughter cell separation, has already been deleted in haploid cells without modify fermentation properties (Marsit et al., 215).
Cas9-induced double-strand break/DNA Repair
HDR
NHEJ
DNA donor template
The Cas (CRISPR-associated) protein combines with an RNA, named guide RNA (gRNA), containing a so-called protospacer sequence at its 5’-end. The protospacer sequence, which specifies DNA cleavage by the Cas endonuclease, is composed of 14-30 bases-long and a short PAM (protospacer adjacent motif) sequence. The repairing of double strand break, generated by Cas9, can be achieved by NHEJ (non-homologous end joining) in the absence of a donor DNA, or by HDR (Homology Directed Repair) when a donor template is present.
CRISPR/Cas9 construction elements
NatMX6-eGFP donor DNA amplified by PCR from pFA6a-TEF2p-eGFP-ADH1- NATMX6 plasmid (Marsit et al, 2015) using 60bp long chimeric primers carrying 40 bp sequence homologous to AMN1 gene targetted by CRISPR/Ca9 system.
Targeting CRISPR/Cas9 system to AMN1 gene
The pRCC-K plasmid was used as vehicle to deliver CRISPR/Cas9 machinery in yeast. To specify the Cas9 break to AMN1, a sgRNA was designed using gRNA Design from ATUM web site (https://www.atum.bio/) and inserted into gRNA scaffold region of pRCC-K using the NEBuilder HiFi DNA Assembly Cloning kit (NEB).
BamHI/HindIII restriction analysis. The loosing of BamHI site verified the presence of gRNA in pEC vector derived from pRCC-K.
NatMX6 TEF2pr eGFP term
SNR52 promoter CYC1 terminator
SV40 NLS SUP4 terminator
gRNA scaffold
pRCC-K 10,255 bp
Donor DNA for Homology-Directed Repair (HDR) to AMN1 locus
To ensure the repairing of the Cas9 DNA double strand break by homologous recombination, a double strand DNA (donor) was co-transformed with the CRISPR-Cas9 plasmid in yeast cells. The donor DNA was composed of the resistance gene for nourseothricin (NatMX6) and the eGFP gene amplified with chimeric primers.
The vector-based delivered of CRISPR/Cas9 pRCC-K (Addgen)
developped by Generoso et al. (2016) and elements of gRNA region.
4
pEC pRCC-K
• •
•
kpb
10 6
Cloning of sgRNA in pRCC-K lead the new pCE plasmid which is able to target a DNA cleavage on AMN1 by the Cas9 endonuclease
WT AMN1::GFP
pEC + NatMX6-eGFP
kbp5 3 2,5 2 1,5 1 0,75 0,5
••• ••
• •
• •
WT C1 C3
Double disruption event sucessfully occured, as the signal for the WT locus disappeared in C3 whereas an off-target effect is observed for C1.
AMN1 AMN1::GFP
S. cerevisiae EC1118 WT (WT) and transformants C1, C2 and C3 observed by confocal microscopy (λex = 475 nm λem = 504 nm). Heterogeneity of GFP expression among individual cells was observed for C1 whereas homogenous but weak signal was noticed for C3.
AMN1 AMN1
eGFP eGFP
CRISPR engineering targeted to AMN1 by co-transformation pEC vector and donor DNA in the diploid EC1118® strain.
C2
100 µmC3
100 µmWT
100 µmC1
100 µmThe CRISPR/Cas9 system successfully GFP-tagged the commercial EC1118® strain by insertion of a GFP gene into AMN1 (C3).
Nevertheless off-target effect was detected (C1) suggesting a non-optimal sgRNA design to target AMN1.
CRISPR engineering of Saccharomyces cerevisiae Lalvin EC1118® strain
Growth of S. cerevisiae EC1118 WT (up) and CRISPRed strain C3 (bottom) in synthetic medium which mimics grape juice. Total cells and cell viability were determined by flow cytometry and Propidium Iodide (PI) labelling. Monitoring of glucose/fructose consumed and ethanol produced was carried out using OenoFoss™.
No modification of oenological capability was detected between CRISPRed strain and parental strain EC1118. CRISPRed yeast C3 presents a stable GFP signal during fermentation.
0 20 40 60 80 100
1E+05 1E+06 1E+07 1E+08 1E+09
0 50 100 150 200 250
Mortality, %
Log (Cells/mL)
Time (h)
EC1118 WT WT live cells/mL % Death WT
0 20 40 60 80 100
1E+05 1E+06 1E+07 1E+08 1E+09
0 50 100 150 200 250
Mortality, %
Log (Cells/mL)
Time (h)
EC1118 AMN1::GFP (C3) fluorescent cells/mL % death C3
In this successful experiment, we used the single plasmid pRCC-K, expressing Cas9 and guide- RNA (Generoso et al., 2016) to insert GFP gene into the Lalvin EC1118® commercial yeast.
CRISPRed yeast didn’t present any modification of oenological characteristics (i.e.
fermentation rate, total alcohol production) compare to parental strain, as expected from this chosen target gene. More optimization, however, is required to obtain better transformation frequencies particularly if this system has to be used with industrial Saccharomyces and non- Saccharomyces strains. Nevertheless, the EC1118 AMN1::GFP C3 strain could be used in future experiments to test yeast-yeast interactions in oenological conditions.
Oenological characterization of CRISPRed yeast
Conclusions
Generation Time G = 5,08 h
Generation Time G = 5,06 h
REFERENCES :
DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., Church, G.M., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343.
Generoso, W.C., Gottardi, M., Oreb, M., Boles, E., 2016. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J. Microbiol. Methods 127, 203–205.
Marsit, S., Mena, A., Bigey, F., Sauvage, F.-X., Couloux, A., Guy, J., Legras, J.-L., Barrio, E., Dequin, S., Galeote, V., 2015. Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts. Mol. Biol. Evol. 32, 1695–1707.
AMN1::GFP AMN1
PCR analysis of AMN1 locus using primers ( ) on both sides of the insertion site.
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