Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 µm and ~0.1 µm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.

Activation of the carbon concentrating mechanism by CO2 deprivation coincides with massive transcriptional restructuring in Chlamydomonas reinhardtii.

A CO(2)-concentrating mechanism (CCM) is essential for the growth of most eukaryotic algae under ambient (392 ppm) and very low (<100 ppm) CO(2) concentrations. In this study, we used replicated deep mRNA sequencing and regulatory network reconstruction to capture a remarkable scope of changes in gene expression that occurs when Chlamydomonas reinhardtii cells are shifted from high to very low levels of CO(2) (≤100 ppm). CCM induction 30 to 180 min post-CO(2) deprivation coincides with statistically significant changes in the expression of an astonishing 38% (5884) of the 15,501 nonoverlapping C. reinhardtii genes. Of these genes, 1088 genes were induced and 3828 genes were downregulated by a log(2) factor of 2. The latter indicate a global reduction in photosynthesis, protein synthesis, and energy-related biochemical pathways. The magnitude of transcriptional rearrangement and its major patterns are robust as analyzed by three different statistical methods. De novo DNA motif discovery revealed new putative binding sites for Myeloid oncogene family transcription factors potentially involved in activating low CO(2)-induced genes. The (CA)(n) repeat (9 ≤ n ≤ 25) is present in 29% of upregulated genes but almost absent from promoters of downregulated genes. These discoveries open many avenues for new research.

The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation.

BACKGROUND: Little is known about the mechanisms of adaptation of life to the extreme environmental conditions encountered in polar regions. Here we present the genome sequence of a unicellular green alga from the division chlorophyta, Coccomyxa subellipsoidea C-169, which we will hereafter refer to as C-169. This is the first eukaryotic microorganism from a polar environment to have its genome sequenced. RESULTS: The 48.8 Mb genome contained in 20 chromosomes exhibits significant synteny conservation with the chromosomes of its relatives Chlorella variabilis and Chlamydomonas reinhardtii. The order of the genes is highly reshuffled within synteny blocks, suggesting that intra-chromosomal rearrangements were more prevalent than inter-chromosomal rearrangements. Remarkably, Zepp retrotransposons occur in clusters of nested elements with strictly one cluster per chromosome probably residing at the centromere. Several protein families overrepresented in C. subellipsoidae include proteins involved in lipid metabolism, transporters, cellulose synthases and short alcohol dehydrogenases. Conversely, C-169 lacks proteins that exist in all other sequenced chlorophytes, including components of the glycosyl phosphatidyl inositol anchoring system, pyruvate phosphate dikinase and the photosystem 1 reaction center subunit N (PsaN). CONCLUSIONS: We suggest that some of these gene losses and gains could have contributed to adaptation to low temperatures. Comparison of these genomic features with the adaptive strategies of psychrophilic microbes suggests that prokaryotes and eukaryotes followed comparable evolutionary routes to adapt to cold environments.