The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

A Novel Non-antibiotic Selectable Marker GASA6 for Plant Transformation

Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure of an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI , which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation.

A novel non-antibiotic selectable marker GASA6 for plant transformation

Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure of an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI, which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation.

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.