Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts.

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids," were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

Seasonal Growth of Zygophyllum dumosum Boiss.: Summer Dormancy Is Associated with Loss of the Permissive Epigenetic Marker Dimethyl H3K4 and Extensive Reduction in Proteins Involved in Basic Cell Functions.

Plants thriving in desert environments are suitable for studying mechanisms for plant survival under extreme seasonal climate variation. We studied epigenetic mechanisms underlying seasonal growth cycles in the desert plant Zygophyllum dumosum Boiss., which was previously shown to be deficient in repressive markers of di-methyl and tri-methyl H3K9 and their association with factors regulating basic cell functions. We showed a contingent association between rainfall and seasonal growth and the epigenetic marker of dimethyl H3K4, which disappears upon entry into the dry season and the acquisition of a dormant state. DNA methylation is not affected by a lack of H3K9 di-methyl and tri-methyl. Changes in methylation can occur between the wet and dry season. Proteome analysis of acid soluble fractions revealed an extensive reduction in ribosomal proteins and in proteins involved in chloroplasts and mitochondrial activities during the dry seasons concomitantly with up-regulation of molecular chaperone HSPs. Our results highlight mechanisms underlying Z. dumosum adaptation to seasonal climate variation. Particularly, summer dormancy is associated with a loss of the permissive epigenetic marker dimethyl H3K4, which might facilitate genome compaction concomitantly with a significant reduction in proteins involved in basic cell functions. HSP chaperones might safeguard the integrity of cell components.

Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica.

The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

Identification and characterization of new Δ-17 fatty acid desaturases.

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54-65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

Isolation of a Δ5 desaturase gene from Euglena gracilis and functional dissection of its HPGG and HDASH motifs.

Delta (Δ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the "desaturation and elongation" pathways. A full length Δ5 desaturase gene from Euglena gracilis (EgΔ5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5' and 3' rapid amplification of cDNA ends. The whole coding region of EgΔ5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that EgΔ5D has about 39 % identity with a Δ5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, EgΔ5D had strong Δ5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of EgΔ5D. A double mutant EgΔ5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type EgΔ5D. Codon optimization of the N-terminal region of EgΔ5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.

Three diacylglycerol acyltransferases contribute to oil biosynthesis and normal growth in Yarrowia lipolytica.

Diacylglycerol (DAG) acyltransferase catalyses the final and committed step of triacylglycerol biosynthesis. Eukaryotes commonly contain up to three distinct classes of DAG acyltransferases: acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), and phospholipid:diacylglycerol acyltransferase (PDAT). The non-conventional oleaginous yeast, Yarrowia lipolytica, contains at least one homologue of each class and serves as a good model to understand the role of different DAG acyltransferases in the biosynthesis of oil, a critical cellular component that serves as a storage molecule as well as a buffer for free fatty acids. We used gene disruptions in Y. lipolytica and in vitro enzyme assays to confirm the identity of genes encoding all three DAG acyltransferases and demonstrate that together they account for almost all oil biosynthesis and that all three contribute significantly to its oil biosynthesis. In Y. lipolytica ATCC 20362 strain, the total lipid% dry cell weight (DCW) as a percentage of the wild-type strain in pdat, dgat1, dgat2, dgat1/dgat2 double mutant and dgat1/dgat2/pdat triple mutant was 70%, 57%, 36%, 18% and 13%, respectively.This is the first example of DGAT1 contributing significantly to oil biosynthesis in a microorganism. The triple mutant shows significant growth defect in both increased lag phase and slower growth rate, suggesting that oil biosynthesis contributes to normal growth in this strain.

Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants.

We report the identification of bifunctional Delta12/omega3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea. The bifunctional activity of these desaturases distinguishes them from all known Delta12 or omega3 fatty acid desaturases. The omega3 desaturase activity of these enzymes also shows a broad omega6 fatty acid substrate specificity by their ability to convert linoleic acid (LA), gamma-linolenic acid, di-homo-gamma-linolenic acid, and arachidonic acid to the omega3 fatty acids, alpha-linolenic acid (ALA), stearidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid (EPA), respectively. Phylogenetic analysis suggests that omega3 desaturases arose by independent gene duplication events from a Delta12 desaturase ancestor. Expression of F. moniliforme Delta12/omega3 desaturase resulted in high ALA content in both Yarrowia lipolytica, an oleaginous yeast naturally deficient in omega3 desaturation, and soybean. In soybean, seed-specific expression resulted in 70.9 weight percent of total fatty acid (%TFA) ALA in a transformed seed compared with 10.9%TFA in a null segregant seed and 53.2%TFA in the current best source of ALA, linseed oil. The ALA/LA ratio in transformed seed was 22.3, a 110- and 7-fold improvement over the null segregant seed and linseed oil, respectively. Thus, these desaturases have potential for producing nutritionally desirable omega3 long-chain polyunsaturated fatty acids, such as EPA, with a significantly improved ratio of omega3/omega6 long-chain polyunsaturated fatty acids in both oilseeds and oleaginous microbes.

Functional dissection of a Rice Dr1/DrAp1 transcriptional repression complex.

We characterized rice cDNA sequences for OsDr1 and OsDrAp1, which encode structural homologs of the eukaryotic general repressors Dr1 and DrAp1, respectively. OsDr1 and OsDrAp1 are nuclear proteins that interact with each other and with the TATA binding protein/DNA complex. In vitro and in vivo functional analyses showed that OsDrAp1 functions as a repressor, unlike its role in other eukaryotic systems, in which DrAp1 is a corepressor. OsDr1 and OsDrAp1 functioned together as a much stronger repressor than either one alone. Functional dissections revealed that the N-terminal histone-fold domains of OsDr1 and OsDrAp1 were necessary and sufficient for their repression and protein-protein interaction with each other. The unique glutamine- and proline-rich domain of OsDr1 had no repression activity. The basic amino acid-rich region and an arginine and glycine repeat domain of OsDrAp1 enhanced its repression activity. Thus, although OsDr1 and OsDrAp1 function as repressors, the functions of the two components are reversed compared with those of their nonplant counterparts.

Progression of geminivirus-induced transgene silencing is associated with transgene methylation.

• The association of viral-induced gene silencing (VIGS) elicited by a DNA virus with DNA methylation of the silenced transgene was studied. • 35S-Green fluorescent protein (GFP) transgenic Nicotiana benthamiana were treated with an inhibitor of DNA methylation, 5-azacytidine (5-Aza-C), and VIGS of the transgene was observed upon inoculation with tomato golden mosaic virus carrying the GFP coding sequence. • The onset of VIGS of the 35S-GFP transgene occurred 14-16 d after inoculation in both control and 5-Aza-C-treated plants. At this stage, the silencing was observed in localized regions. Silencing was uniform by 30 d after inoculation in plants that had methylated GFP-DNA, whereas plants that continued to display the same phenotype as seen at 14-15 d after inoculation had hypomethylated GFP-DNA. Viral expression of GFP persisted in pockets throughout the life of infected plants. • This is the first demonstration of a correlation between post transcriptional gene silencing induced by a DNA virus, and transgene methylation. The results suggest that, while DNA methylation is not necessary for the initiation of silencing, the progression of silencing is affected by inhibition of DNA methylation.