CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.
A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Robert
R
Mans
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Harmen M
HM
van Rossum
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Melanie
M
Wijsman
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Antoon
A
Backx
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Niels G A
NG
Kuijpers
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Marcel
M
van den Broek
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Pascale
P
Daran-Lapujade
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Jack T
JT
Pronk
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Antonius J A
AJ
van Maris
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands J.G.Daran@TUDelft.nl.
Jean-Marc G
JM
Daran
D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004720:Endonucleases
CRISPR/Cas9; S. cerevisiae, gRNA; genetic modification; plasmid; webtool