CRISPR/Cas9 advances engineering of microbial cell factories

One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering

CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae

BACKGROUND: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers. RESULTS: Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. CONCLUSIONS: The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0288-3) contains supplementary material, which is available to authorized users

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain

Modular 5'-UTR hexamers for context-independent tuning of protein expression in eukaryotes.

Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes