Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects

Abstract

Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.

Yeast genetic interaction screens in the age of CRISPR/Cas

“…6a). There are several excellent reviews on the discovery and mechanism of CRISPR/Cas (Lander 2016; Stovicek et al 2017; Raschmanová et al 2018). …”

“…There are over two dozen CRISPR/Cas toolkits for S. cerevisiae , most of which have been described in detail by Stovicek et al (2017) and Raschmanová et al (2018). Most CRISPR/Cas editing tools combine the naturally separate CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) in a hybrid small guide RNA (sgRNA).…”

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

“…Therefore, the sgRNA comprises a structural sequence for Cas9 binding and a variable 20 bp sequence to match the genomic target sequence. In addition, a donor DNA (dDNA) containing the engineered genomic target with homology flanks must be provided for the DSB repair by HR (Jinek et al ., ; Raschmanová et al ., ).…”

“…Within the last few years, the Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas system (CRISPR/Cas9) has become an efficient and marker-less approach for genome editing in yeast and filamentous fungi (Shi et al, 2017;Raschmanov a et al, 2018). In the model yeast S. cerevisiae, an extensive toolbox of CRISPR-related applications has been developed since the yeast system was first described (Dicarlo et al, 2013;Raschmanov a et al, 2018). Therefore, the development of a marker-less CRISPR/Cas9 method for genomic engineering of A. gossypii represents an important challenge willing to expand the molecular toolbox of this industrial fungus.…”

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

“…Despite the increasing interest towards this species, Z. rouxii remains underexploited in industrial and food bioprocesses due to the low availability of synthetic biology tools, often required to enhance yield, titre, and production rate also in strains with beneficial native phenotypes. In recent years, the type II CRISPR‐Cas9 system from Streptococcus pyogenes has been first adopted in genome editing of S. cerevisiae and then species specifically adapted for editing some nonconventional yeasts, such as Y. lipolytica , P. pastoris , K. lactis , Candida albicans , and Candida glabrata (Raschmanova, Weninger, Glieder, Kovar, & Vogl, ). This species‐specific implementation highly benefited from the extensive repertoire of synthetic tools and engineering know‐out accumulated in the past decades, since high efficient and specific CRISPR‐Cas9 mutagenesis often involves an extensive multistep optimisation to identify suitable selective makers, develop high efficient transformation protocols and gain appropriate nuclear delivery and regulated expression of the sgRNAs and Cas9 protein (Wang et al, ).…”