A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity

Macarena Larroude,Heykel Trabelsi,Jean-Marc Nicaud,Tristan Rossignol

doi:10.1007/s10529-020-02805-4

Abstract

ObjectivesThe construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited, including those of wild-type strains.ResultsUsing the Golden Gate method, we assembled a set of six CRISPR/Cas9 vectors, each containing a different selection marker, to be used for editing the genome of the industrial yeast Y. lipolytica. This vector set is available via Addgene. Any guide RNA (gRNA) sequence can be easily and rapidly introduced in any of these vectors using Golden Gate assembly. We successfully edited six different genes in a variety of genetic backgrounds, including those of wild-type strains, with five of the six vectors. Use of these vectors strongly improved homologous recombination and cassette integration at a specific locus.ConclusionsWe have created a versatile and modular set of CRISPR/Cas9 vectors that will allow any Y. lipolytica strain to be rapidly edited; this tool will facilitate experimentation with any prototroph wild-type strains displaying interesting features.

Highlights

Yarrowia lipolytica is widely used as a microbial cell factory chassis in the development of industrial applications aiming to produce fatty acids, organic acids, or enzymes (Nicaud 2012; Madzak 2015; Ledesma-Amaro and Nicaud 2016)
A guide RNA system involving the expression of orthogonal T7 polymerase was exported to Y. lipolytica; it was based on the pCRISPRyl vector of Schwartz et al (Morse et al 2018)
Our design was based on the basal structure of the plasmid developed by Schwartz et al (Schwartz et al 2016), and the Golden Gate method made it possible to assemble and switch out different parts

Summary

Introduction

Yarrowia lipolytica is widely used as a microbial cell factory chassis in the development of industrial applications aiming to produce fatty acids, organic acids, or enzymes (Nicaud 2012; Madzak 2015; Ledesma-Amaro and Nicaud 2016). CRISPR/Cas technology is being continually refined, and it was rapidly implemented in Saccharomyces cerevisiae (DiCarlo et al 2013). It has been successfully used in Y. lipolytica, with the aim of overcoming the aforementioned limitations and accelerating engineering cycles. Several CRISPR/Cas systems dedicated to Y. lipolytica have recently been described. Both an RNA polymerase II (Pol II) transcription system and RNA polymerase III (Pol III) elements have been set up. Schwartz et al (Schwartz et al 2016) used a Pol III system and developed an efficient synthetic Pol III-tRNA hybrid promoter for gene disruption. Alternative systems, such as using defective Cas (CRISPRi) to inhibit expression, CRISPRa to activate gene expression, and the dual CRISPR/Cas system to excise and integrate genes have been shown to function in Y. lipolytica (Schwartz et al 2017a, 2018; Gao et al 2018)

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Conclusion