Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, <i>Nannochloropsis oceanica</i> CCMP1779

Poliner, Eric; Takeuchi, Tomomi; Du, Zhi‐Yan; Benning, Christoph; Farré, Eva M.

doi:10.1021/acssynbio.7b00362

Cited by 110 publications

(156 citation statements)

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“…FLUC has been widely used as a circadian reporter in multiple systems (Welsh et al , 2005, Welsh and Kay 2005), however, RLUC and the engineered NLUC have the advantage of being smaller proteins, and catalyze ATP-independent reactions (England et al , 2016, Hall et al , 2012). We have recently used NLUC to quantify protein expression in N. oceanica CCMP1779 (Poliner et al , 2018c). The codon optimized luciferase coding regions were expressed under the control of the CELLULOSE SYNTHASE promoter ( CS ), a gene with strong oscillations in RNA levels under light/dark cycles (Poliner et al , 2015).…”

Section: Resultsmentioning

confidence: 99%

“…Several Nannochloropsis strains from different worldwide locations have been sequenced and current data suggests that Nannochloropsis species are haploid with genome sizes of ~30 Mb, containing from ~6,500 to ~12,000 genes (Corteggiani Carpinelli et al , 2014, Radakovits et al , 2012, Schwartz et al , 2018, Vieler et al , 2012, Wang et al , 2014). Recent years have seen the development of a comprehensive set of molecular tools for working with these algae, such as homologous gene replacement, overexpression, gene silencing and CRISPR/Cas9 based genome editing (Kilian et al , 2011, Poliner et al , 2018a, Poliner et al , 2018b, Poliner et al , 2018c, Verruto et al , 2018, Wang et al , 2016, Wei et al , 2017). In this study, we describe the development of a bioluminescence reporter system for the long-term in vivo measurement of gene expression in two Nannochloropsis species.…”

Section: Introductionmentioning

confidence: 99%

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Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

Poliner

Cummings

Newton

et al. 2019

Preprint

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32Circadian clocks allow organisms to predict environmental changes caused by the rotation of the 33 Earth. Although circadian rhythms are widespread among different taxa, the core components of 34 circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. 35Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly 36 characterized and no clock components have been identified. We have investigated cell division 37 and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division 38 occurred principally during the night period under diel conditions, however, rhythms dampened 39 within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for 40 long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, N. 41 oceanica CCMP1779 and N. salina CCMP537. The reporter lines express free-running 42 bioluminescence rhythms with periods of ~21-31 h that dampen within ~3-4 days under constant 43 light. Using different entrainment regimes, we demonstrate that these rhythms are regulated by a 44 circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be 45 modulated pharmacologically using a CK1 e/d inhibitor, suggesting a role of this kinase in the 46 Nannochloropsis clock. Together with the molecular and genomic tools available for 47 Nannochloropsis species, these reporter lines represent an excellent system for future studies on 48 the molecular mechanisms of stramenopile circadian oscillators. 49 50 Significance statement 51Stramenopiles are a large and diverse line of eukaryotes in which circadian rhythms have been 52 only poorly characterized and no clock components have been identified. We have developed 53 bioluminescence reporter lines in Nannochloropsis species and provide evidence for the presence 54 of a circadian oscillator in stramenopiles; these lines will serve as tools for future studies to 55 uncover the molecular mechanisms of circadian oscillations in these species. 56 57

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

Poliner

Cummings

Newton

et al. 2019

Preprint

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“…Nannochloropsis are important industrial microalgae due to their robust growth and high lipid contents, and extensive efforts are dedicated to genetic improvements (Ma, Chen, Yang, Liu, & Chen, ; Poliner, Takeuchi, Du, Benning, & Farre, ; Radakovits et al, ). In particular, TF engineering has been used in Nannochloropsis for improving biomass and/or lipid production (Ajjawi et al, ; Kang et al, ; Kwon et al, ); however, these have been achieved in the batch culture in a laboratory scale, and lack assessment of potential for industrial production.…”

Section: Discussionmentioning

confidence: 99%

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor

Kang

Kim

Sung

et al. 2019

Biotech & Bioengineering

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Microalgae are promising feedstocks for sustainable and eco‐friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix‐loop‐helix transcription factor (NsbHLH2). These transformants showed an improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize the growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild‐type, NsbHLH2 transformant consumed more nitrate at a high dilution rate (0.5 day −1 ), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l −1 day −1 . To elucidate genetic mechanisms, we identified the genes containing E‐boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real‐time polymerase chain reaction. These results provide proof‐of‐concept that NsbHLH2 can be used to produce biomass and lipids.

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“…FLUC has been widely used as a circadian reporter in multiple systems , but RLUC and the engineered NLUC have the advantage of being smaller proteins, and catalyze ATP-independent reactions (Hall et al, 2012;England et al, 2016). We have recently used NLUC to quantify protein expression in N. oceanica CCMP1779 (Poliner et al, 2018c). The codon-optimized luciferase coding regions were expressed under the control of the cellulose synthase promoter (CS), a gene with strong oscillations in RNA levels under light/dark cycles (Poliner et al, 2015).…”

Section: Development Of Luciferase Reporter Lines In Nannochloropsis mentioning

confidence: 99%

“…Promoter reporter vectors were linearized by restriction digest, concentrated by ethanol precipitation, and resuspended in water. Transformations were conducted as described previously (Poliner et al, 2018c).…”

Section: Generation Of Luciferase Expressing Linesmentioning

confidence: 99%

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

et al. 2019

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Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free non-transgenic engineered lines. To test this system, we disrupted the nitrate reductase gene (NR) and subsequently removed the CRISPR episome to generate non-transgenic marker-free nitrate reductase knockout lines (NR-KO).

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Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779

Cited by 110 publications

References 34 publications

Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

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