A CRISPR/Cas9 method facilitates efficient oligo-mediated gene editing in Debaryomyces hansenii

Abstract

The halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques does not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism pose further challenges when precise gene targeting is required. In this study, we present a novel plasmid based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple sgRNAs that provides possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed an NHEJ deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single stranded DNA oligonucleotides are sufficient to direct repair of DNA breaks induced by sgRNA-Cas9 resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeastCandida intermedia

“…First, we set out to identify a working transformation protocol and a functional selectable marker gene for C. intermedia . Like many other CTG clade yeast (Vyas et al 2015 , Norton et al 2017 , Ennis et al 2021 , Strucko et al 2021 ), C. intermedia displayed sensitivity to cloNAT in the concentration of 10–1000 µg/ml (data not shown), of which 200 µg/ml was chosen in further experiments. We transformed C. intermedia using electroporation and a linearized CaNAT1 marker cassette that has proven effective in numerous yeast including C. albicans, C. parapsilosis , and C. intermedia ’s close relative Clavispora lusitaniae (outlined in Fig.…”

“…Our analysis showed that C. intermedia is a haploid yeast belonging to the CTG clade of ascomycetous yeast, where the universal CUG codon is predominantly translated into a serine rather than a leucine (Geijer et al 2020 , Muhlhausen et al 2021 ). The CTG clade comprises many relatively well-studied yeasts including human pathogens Candida albicans, Candida tropicalis, Candida parapsilosis , and industrially attractive yeasts such as Debaryomyces hansenii, Metschnikowia pulcherrima , and Scheffersomyces stipitis (Enkler et al 2016 , Cao et al 2018 , Muhlhausen et al 2021 , Strucko et al 2021 , Zhang et al 2022 ). Genome editing tools have been developed for many of these species, which means that several CTG clade-compatible plasmids containing selectable marker genes and reporter systems are available (Papon et al 2012 , Lobs et al 2017 , Uthayakumar et al 2021 ).…”

“…Another, more recently developed solution for markerless, multiple genome editing is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) (Jinek et al 2012 ). Although the list of species that can be modified by this method expands every year, development of CRISPR/Cas9-based tools for new yeast species is not trivial and often requires species-specific optimization of gene expression, transformation, and selection protocols (Vyas et al 2015 , Ng and Dean 2017 , Ennis et al 2021 , Strucko et al 2021 ).…”

Utilization of salt‐rich by‐products from the dairy industry as feedstock for recombinant protein production by Debaryomyces hansenii

“…YFP has been chosen in this work and has been cloned into the yeast by random integration. Although CRISPR/Cas9 tools to genetically modify D. hansenii are now available (Strucko et al, 2021 ), it is still unknown which integration sites in the genome of this yeast are the most suitable to introduce a recombinant gene for optimal expression. Hence, random integration has been chosen as an alternative in this study.…”

“…The transformation was performed following the electroporation protocol described by Strucko et al ( 2021 ), using the MicroPulser electroporator (BioRad) on manual settings at 2.3 kV. YPD + NTC agar plates were used for the selection of the transformant cells.…”