Won-Joong Jeong

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.

Droplet electroporation in microfluidics for efficient cell transformation with or without cell wall removal

An efficient cell transformation method is presented that utilizes droplet electroporation on a microfluidic chip. Two types of green microalgae, a wall-less mutant and a wild type of Chlamydomonas reinhardtii, are used as model cells. The PDMS-glass electroporation chip is simply composed of a flow-focusing microstructure for generating cell-encapsulating droplets and a serpentine channel for better mixing of the content in the droplet, and five pairs of parallel microelectrodes on the glass slide, without involving any expensive electrical equipment. The transformation efficiency via the microfluidic electroporation is shown to be more than three orders of magnitude higher for the wall-less mutant, and more than two orders of magnitude higher for the wild type, which has its cell wall intact, than bulk phase electroporation under identical conditions. Furthermore, the microfluidic transformation is remarkably efficient even at a low DNA/cell ratio, facilitating ways of controlling the transgenic copy number, which is important for the stability of the transgene expression.

Plastid and mitochondrion genomic sequences from Arctic Chlorella sp. ArM0029B

BackgroundChorella is the representative taxon of Chlorellales in Trebouxiophyceae, and its chloroplast (cp) genomic information has been thought to depend only on studies concerning Chlorella vulgaris and GenBank information of C. variablis. Mitochondrial (mt) genomic information regarding Chlorella is currently unavailable. To elucidate the evolution of organelle genomes and genetic information of Chlorella, we have sequenced and characterized the cp and mt genomes of Arctic Chlorella sp. ArM0029B.ResultsThe 119,989-bp cp genome lacking inverted repeats and 65,049-bp mt genome were sequenced. The ArM0029B cp genome contains 114 conserved genes, including 32 tRNA genes, 3 rRNA genes, and 79 genes encoding proteins. Chlorella cp genomes are highly rearranged except for a Chlorella-specific six-gene cluster, and the ArM0029B plastid resembles that of Chlorella variabilis except for a 15-kb gene cluster inversion. In the mt genome, 62 conserved genes, including 27 tRNA genes, 3 rRNA genes, and 32 genes encoding proteins were determined. The mt genome of ArM0029B is similar to that of the non-photosynthetic species Prototheca and Heicosporidium. The ArM0029B mt genome contains a group I intron, with an ORF containing two LAGLIDADG motifs, in cox1. The intronic ORF is shared by C. vulgaris and Prototheca. The phylogeny of the plastid genome reveals that ArM0029B showed a close relationship of Chlorella to Parachlorella and Oocystis within Chlorellales. The distribution of the cox1 intron at 721 support membership in the order Chlorellales. Mitochondrial phylogenomic analyses, however, indicated that ArM0029B shows a greater affinity to MX-AZ01 and Coccomyxa than to the Helicosporidium-Prototheca clade, although the detailed phylogenetic relationships among the three taxa remain to be resolved.ConclusionsThe plastid genome of ArM0029B is similar to that of C. variabilis. The mt sequence of ArM0029B is the first genome to be reported for Chlorella. Chloroplast genome phylogeny supports monophyly of the seven investigated members of Chlorellales. The presence of the cox1 intron at 721 in all four investigated Chlorellales taxa indicates that the cox1 intron had been introduced in early Chorellales as a cis-splice form and that the cis-splicing intron was inherited to recent Chlorellales and was recently trans-spliced in Helicosporidium.

Heat shock protein gene family of the Porphyra seriata and enhancement of heat stress tolerance by PsHSP70 in Chlamydomonas.

Heat shock proteins and molecular chaperones are key components contributing to survival in the abiotic stress response. Porphyra seriata grows on intertidal rocks exposed to dynamic environmental changes associated with the turning tides, including desiccation and heat stress. Analysis of the ESTs of P. seriata allows us to identify the nine HSP cDNAs, which are predicted to be PsHSP90, three PsHSP70, PsHSP40 and PsHSP20, and three 5′-truncated HSP cDNAs. RT–PCR results show that most of the PsHSP transcripts were detected under normal cell growth conditions as well as heat stress, with the exception of two cDNAs. In particular, PsHSP70b and PsHSP20 transcripts were upregulated by heat stress. When the putative mitochondrial PsHSP70b was introduced and overexpressed in Chlamydomonas, transformed Chlamydomonas evidenced higher rates of survival and growth than those of the wild type under heat stress conditions. Constitutive overexpression of the PsHSP70b gene increases the transcription of the HSF1 as well as the CrHSP20 and CrHSP70 gene. These results indicate that PsHSP70b is involved in tolerance to heat stress and the effects on transcription of the CrHSP20 and CrHSP70 genes.

A new Arctic Chlorella species for biodiesel production

Microalgae are a potential resource for biodiesel production. A green alga, Chlorella sp., was isolated from Arctic sea ice, which was named ArM0029B. These algae displayed faster growth at a wide temperature range of 4-32°C compared to Chlorella vulgaris. ArM0029B also accumulated high levels of total fatty acids under nitrogen starvation conditions, reaching 39% of dry cell weight, with the proportion of oleic acid (18:1) and linoleic acid (18:2) reaching 54% of total fatty acids. Taken together, these results indicate that the newly identified Chlorella species, ArM0029B, is a promising candidate for biodiesel production.

IDENTIFICATION OF THE HIGH-TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.

Production of human lactoferrin in transgenic cell suspension cultures of sweet potato

Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred to Murashige and Skoog (MS) medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm−3 kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were established in liquid MS medium with 4.52 µM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up to 3.2 µg mg−1 (total protein).

Comparative proteomics using lipid over-producing or less-producing mutants unravels lipid metabolisms in Chlamydomonas reinhardtii

In this study, proteomic approach was employed to advance our understanding of genes associated with microalgal lipid production. We first generated random mutants of Chlamydomonas reinhardtii, which displayed increased or decreased lipid content, compared to that in the wild-type. Mutants were designated as CR12 and CR48. Then, the changes of protein expression associated with the mutations of Chlamydomonas reinhardtii were analyzed. We could identify proteins that were significantly up-regulated or down-regulated either in the wild-type or CR12 or CR48. Our results will help understand additional genes or pathways directly or indirectly linked to microalgal lipid production.

Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal

A unique digital microfluidic electroporation (EP) system successfully demonstrates higher transgene expression than that of conventional techniques, in addition to reliable productivity and feasible integrated processes. By systematic investigations into the effects of the droplet EP conditions for a wild-type microalgae, 1 order of magnitude higher transgene expression is accomplished without cell wall removal over the conventional bulk EP system. In addition, the newly proposed droplet EP method by a droplet contact charging phenomena shows a great potential for the integration of EP processes and on-chip cell culture providing easy controllability of each process. Finally, the implications of the accomplishments and future directions for development of the proposed technology are discussed.

Cucumber mosaic virus 2b protein inhibits RNA silencing pathways in green alga Chlamydomonas reinhardtii

The functions of RNA silencing are repression of endogenous gene expression and antiviral defense in plants and animals. Cucumber mosaic virus 2b (CMV2b) is a suppressor of RNA silencing in higher plants. In the present study, we evaluated the RNA silencing suppressor activity of CMV2b in Chlamydomonas reinhardtii. Before transformation, we modified CMV2b codons to increase the GC content for optimal expression in C. reinhardtii. Inhibition of Maa7 silencing was detected in CMV2b-expressing Maa7-IR44 strains, indicating that CMV2b suppressed siRNA pathways in C. reinhardtii as in higher plants. In addition, mRNA expression targeted for cleavage by miRNA was significantly higher in CMV2b-expressing strains, but increased accumulation of miRNA was not detected. These results indicate that the suppression of miRNA pathways is mediated by CMV2b in C. reinhardtii. Interestingly, expression of both Argonaute 1 (AGO1) and Dicer-like 1 (DCL1), regulated by a bidirectional promoter, was reduced in CMV2b-expressing strains, suggesting that CMV2b may affect transcription factors involved in RNA silencing pathways. Furthermore, reduction of AGO2 and AGO3 expression was detected in CMV2b-expressing strains. Taken together, our results demonstrate that CMV2b may suppress both siRNA and miRNA pathways, and also impair AGOs and DCL1 expression in C. reinhardtii.