Abstract
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.
Highlights
Saccharomyces cerevisiae has been successfully used as a model organism to decipher biological processes in higher eukaryotes (Botstein and Fink 2011) and as a popular metabolic engineering platform (Nielsen et al 2013)
Where previous studies mainly focused on the use of Clustered Regularly Interspaced Short Palindromic Repeats (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
Because the CRISPR/Cas9 system can be highly sequence specific, it is crucially important to select target sequences based on correct reference genome sequence information
Summary
Saccharomyces cerevisiae has been successfully used as a model organism to decipher biological processes in higher eukaryotes (Botstein and Fink 2011) and as a popular metabolic engineering platform (Nielsen et al 2013). Bacteria have developed several systems to degrade foreign DNA Very quickly after their discovery, restriction enzymes became the ‘workhorses of molecular biology’ (reviewed by Roberts 2005). The typeII bacterial CRISPR system of Streptococcus pyogenes requires the Cas nuclease and the RNA complex that guides it to a specific sequence of the (foreign) DNA. This RNA complex generally consists of two RNA molecules: the CRISPR RNA (crRNA) and the trans-activating CRISPR RNA (tracrRNA). Cas9-based systems have been used for the construction of (multiplexed) genetic modifications in a variety of organisms, including human pluripotent stem cells (Gonzalez et al 2014), zebrafish (Hwang et al 2013), plants (Feng et al 2013), flies (Gratz et al 2013) and mice (Wang et al 2013; for a more extensive list, see Hsu, Lander and Zhang 2014)
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