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WO2022189976A1 - Altérations génétiques dans des organismes microalgal, procédés et compositions - Google Patents

Altérations génétiques dans des organismes microalgal, procédés et compositions Download PDF

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WO2022189976A1
WO2022189976A1 PCT/IB2022/052064 IB2022052064W WO2022189976A1 WO 2022189976 A1 WO2022189976 A1 WO 2022189976A1 IB 2022052064 W IB2022052064 W IB 2022052064W WO 2022189976 A1 WO2022189976 A1 WO 2022189976A1
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euglena
organism
gene
nucleic acid
acid molecule
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Zhiyong Zhang
Scott Farrow
Adam J. NOBLE
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Noblegen Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)

Definitions

  • the present disclosure relates to a microalgae organism with a non- transgenic, human-induced, genetic alteration, a method of altering a trait in a microalgae organism through non-transgenic, human-induced, genetic alteration, a method of expressing a heterologous nucleic acid molecule in a target genome of a microalgae organism, a composition for expressing a heterologous nucleic acid molecule in a target genome site of a microalgae organism, a method of expressing a heterologous nucleotide sequence in a microalgae organism, and a method of producing proteins, lipids, amino acids, fatty acids, or combinations thereof by culturing a microalgae organism.
  • Microalgae are a rich source of protein, essential fatty acids, vitamins, and minerals. After lipid removal, the residual biomass contains even higher concentrations of protein and other nutrients. Microalgae are good sources of long chain polyunsaturated fatty acids (“PUFA”) and have been used to enrich diets with omega-3 PUFAs.
  • PUFA long chain polyunsaturated fatty acids
  • a type of microalgae named Euglena belongs to a group of single-celled microscopic algae, that is often used as a candidate species for laboratory studies and technological applications.
  • Species of the microalgae genus Euglena offer several advantages as sources of rich nutrients and other meaningful natural products, including a flexible metabolism with remarkable metabolic capacity that can be leveraged for the production of numerous chemical scaffolds under a wide variety of culture conditions. This includes growth modalities (/. e. , mixotrophy, autotrophy, and heterotrophy) that facilitate its culture on a variety of carbon/nitrogen sources and in classically inhospitable environments ( e.g ., extreme pH and heavy metal tolerance), scalability, advanced post- translational modification machinery, and ease of cultivation. Furthermore, microalgae of the genus Euglena naturally produce large quantities of vitamins, proteins, wax esters, lipids, and products with immune stimulating activities like paramylon, making it an attractive microbe for the production of high-value natural products.
  • One aspect of the present disclosure relates to a Euglena organism comprising a non-transgenic, human-induced alteration in the GSL2 gene.
  • the Euglena organism also has a property selected from the group consisting of (i) reduced carbohydrate content, (ii) reduced size of paramylon granules, (iii) increased protein content, (iv) increased lipid content, (v) altered amino acid profile, (vi) altered fatty acid profile, and (vii) any combination thereof compared to a wild type Euglena organism of a same species comprising a GSL2 gene.
  • Another aspect of the present disclosure relates to a method of altering a trait in a Euglena organism.
  • This method involves providing a ribonuclear protein (RNP) complex comprising a guide RNA having complementarity to an endogenous gene of a Euglena organism and a Clustered Regularly Interspaced Short Palindromic Repeats (“CR1SPR”) Cas-related nuclease.
  • RNP ribonuclear protein
  • C1SPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the method further involves selecting for the genetic alteration in the organism, where the organism comprises the genetic alteration and a property selected from the group consisting of (i) reduced carbohydrate content, (ii) increased protein content, (iii) increased lipid content, (iv) altered amino acid profile, (v) altered fatty acid profile, (vi) reduced or underdeveloped chloroplasts, and (vii) any combination thereof as compared to a wild type Euglena organism of a same species.
  • a further aspect of the present disclosure relates to a method of expressing a heterologous nucleic acid molecule in a target genome insertion site of a Euglena organism.
  • This method involves providing an RNP complex comprising a guide RNA having complementarity to an endogenous gene of a Euglena organism and a CRISPR Cas-related nuclease.
  • a single stranded oligodeoxynucleotide (ssODN) is provided, where the ssODN comprises a first homology arm, a heterologous nucleic acid molecule, and a second homology arm, where the first and second homology arms have complementarity to regions flanking the target genome insertion site.
  • the method also involves introducing the RNP complex and the ssODN into a Euglena organism, where the heterologous nucleic acid molecule is inserted into the target genome insertion site and is expressed.
  • compositions for expressing a heterologous nucleic acid molecule in a target genome insertion site of a Euglena organism comprises an ssODN, where the ssODN comprises a first homology arm, a heterologous nucleic acid molecule, and a second homology arm, where the first and second homology arms have complementarity to regions flanking the target genome insertion site.
  • the composition also comprises a guide RNA having complementarity to an endogenous gene of a Euglena organism and a CRISPR Cas- related nuclease, where the Cas-related nuclease cleaves the target genome insertion site and where the nucleic acid molecule encoding the heterologous nucleic acid molecule is inserted into the target genome insertion site.
  • a further aspect of the present disclosure relates to a method of expressing a heterologous nucleic acid molecule in a Euglena gracilis strain Z, line B2 organism.
  • This method involves transforming a Euglena gracilis strain Z, line B2 organism with a DNA vector comprising a heterologous nucleic acid molecule, a promoter operably linked to the heterologous nucleic acid molecule, and a selectable marker, where said transforming expresses the heterologous nucleic acid molecule in the organism.
  • a further aspect of the present disclosure relates to a method of producing proteins, lipids, amino acids, fatty acids, or combinations thereof comprising culturing a Euglena organism that does not express biologically functional glucan synthase-like 2 (GSL2) protein in a medium and harvesting the proteins, lipids, amino acids, fatty acids, or combinations thereof.
  • GSL2 biologically functional glucan synthase-like 2
  • Described herein is the development of genetic engineering methods, compositions, and novel cell lines to produce high value-added products from microalgae.
  • Novel microalgae with high levels of protein, high levels of lipids, and low levels of carbohydrates were developed using ribonucleoprotein complex (“RNP”) delivered CRISPR/Cas-related nucleases to alter specific microalgae genes.
  • RNP ribonucleoprotein complex
  • Eg-GSL2 paramylon synthase gene
  • B2 and B3 were both paramylon- free, and B2 was also chloroplast-free, while B3 had underdeveloped chloroplasts.
  • CK wild type check
  • Euglena gracilis strain Z Euglena gracilis lines B2 and B3 exhibited unique properties including enhanced production of proteins and lipids, and reduced carbohydrates. Specific amino acids and fatty acids were also markedly different in both B2 and B3 compared to wild type.
  • GFP green- fluorescent protein
  • a nucleic acid molecule for the gene encoding hygromycin resistance (HygR) gene hygromycin phosphotransferase (Hptll) was successfully introduced in-frame and directly under the endogenous gene expression regulatory elements (both promoter and terminator) of the paramylon degradation gene, Egcell 7 A, in wild type Euglena gracilis for the heterologous production of the hygromycin phosphotransferase.
  • FIG. 1 is a graph of Euglena gracilis gene expression levels and verification of the active paramylon synthase candidate gene ⁇ - 1 ,3-glucan synthase-like 2 (“G5Z2”) over time.
  • the time points were analyzed in days post inoculation (“DPI”), including time 0 (“T0-0DPI”), 2 days (“T1-2DPI”), 4 days (“T2-4DPI”), 7 days (“T3- 7DPI”), and 14 days (“T4-14DPI”). Transcription from the GSL2 gene was detected, but not from its homolog, ⁇ - 1 ,3-glucan synthase-like 1 'GSLP”) under heterotrophic/aerobic growth conditions.
  • DPI days post inoculation
  • T0-0DPI time 0
  • T1-2DPI 2 days
  • T2-4DPI 4 days
  • T3- 7DPI 7 days
  • T4-14DPI 14 days
  • trans-2-enoyl-coA reductase (“T2R-1”), delta-9 elongase (“d9E-l”), delta-8 desaturase gene 1 (“d8Dl-l”), delta-8 desaturase gene 2 (“d8D2-2”), delta-5 desaturase (“d5D-l”), and delta-4 desaturase (“d4D-l”); wax ester genes fatty acyl-coenzyme A reductase (“FAR1”) and wax synthase (“WS1”); and paramylon genes, b-1,3 ⁇ E ⁇ 03h synthase-like 1 (“GSLl-1”) and b-1,3 ⁇ E ⁇ 03h synthase like-2 (“GSL2-1”).
  • FIG. 2 is an allelic discrimination plot using gene-specific markers that were developed to detect the genome edited Eg-gsl2-mt allele with the 6 bp nucleotide deletion within the GSL2 gene in the genomes of the B2 and B3 cell lines (“ Eg-gsl2-mt - crP2”) compared to the wild type GSL2 allele (“Eg-GSL2-WT-crPl”).
  • Eg-gsl2-mt - crP2 the wild type GSL2 allele
  • Eg-GSL2-WT-crPl wild type GSL2 allele
  • the plot shows genotyping of homozygous Eg-GSL2- WT-crPl wild type (WT) alleles in the lower right comer, and homozygous Eg-gsl2-mt- crP2 alleles in the upper left comer of the plot.
  • the markers can also be used to detect heterozygous Eg-GLS2-WT/Eg-gsl2-mt lines. Squares indicate negative controls.
  • FIG. 3 is a graph of paramylon synthase ( GSL2 ) gene’s relative expression results over time (days 0, 1, 2, 3, and 4) during heterotrophic/aerobic growth.
  • the graph showed that the CRISPR modified paramylon- free B2 cell line had significantly reduced GSL2 transcription levels at all the time points during the heterotrophic/aerobic culture conditions.
  • the 6 bp nucleotide deletion in GSL2 did not abolish transcription of mRNA from the gene, but the Eg-gsl2 -edited allele of the GSL2 gene was no longer functional for paramylon synthesis.
  • FIG. 4 is a bar graph of the total amino acid content in B2, B3, WT cell lines under various culture conditions: heterotrophic and aerobic (HA), heterotrophic and anaerobic (HN), mixotrophic and aerobic (MA) and mixotrophic and anaerobic (MN) for B2, B3, and WT cell lines. Error bars represent standard error.
  • FIG. 5 is a bar graph of the amino acid profile of B2, B3, and WT cell lines under heterotrophic and aerobic growth condition. Amino acid profiles are reported as mass of the given amino acid (g) per lOOg of the dried biomass. The 9 essential amino acids are group together on the far right of the figure. Error bars represent standard error.
  • FIG. 6 is a bar graph of the amino acid profile of B2, B3, and WT cell lines under heterotrophic and anaerobic growth condition. Amino acid profiles are reported as mass of the given amino acid (g) per lOOg of the dried biomass. The 9 essential amino acids are group together on the far right of the figure. Error bars represent standard error.
  • FIG. 7 is a bar graph of the amino acid profile of B2, B3, and WT cell lines under mixotrophic and aerobic growth condition. Amino acid profiles are reported as mass of the given amino acid (g) per lOOg of the dried biomass. The 9 essential amino acids are group together on the far right of the figure. Error bars represent standard error.
  • FIG. 8 is a bar graph of the amino acid profile of B2, B3, and WT cell lines under mixotrophic and anaerobic growth condition. Amino acid profiles are reported as mass of the given amino acid (g) per lOOg of the dried biomass. The 9 essential amino acids are group together on the far right of the figure. Error bars represent standard error.
  • FIG. 9 is a bar graph of myristic, palmitic, oleic, dihomo-gamma-linolenic fatty acids of B2, B3, and WT under heterotrophic aerobic, heterotrophic anaerobic, mixotrophic aerobic, and mixotrophic anaerobic conditions. Faty acids are presented as mg of faty acid per g of dried biomass. Error bars represent standard error.
  • FIG. 10 is a bar graph of tridecylic and tricosylic fatty acids of B2, B3, and WT under heterotrophic aerobic, heterotrophic anaerobic, mixotrophic aerobic, and mixotrophic anaerobic conditions. Faty acids are presented as mg of faty acid per g of dried biomass. Error bars represent standard error.
  • FIG. 11 is a bar graph of the relative gene expression of the paramylon synthase gene, Eg-GSL2 under mixotrophic & aerobic growth condition. Relative expression of the Eg-GSL2 gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 12 is a bar graph of the relative gene expression of the chloroplast gene, Chl-ccsA under mixotrophic & aerobic growth condition. Relative expression of the Chl-ccsA gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 13 is a bar graph of the relative gene expression of the glycolysis gene, Eg-GAPDH/Eg-GapC under mixotrophic & aerobic growth condition. Relative expression of the Eg-GAPDH/Eg-GapC gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 14 is a bar graph of the relative gene expression of the paramylon degradation gene, Egcell 7 A under mixotrophic & aerobic growth condition. Relative expression of the Egcell 7 A gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 15 is a bar graph of the relative gene expression of the dark specific gene, Eg-PNO under mixotrophic & aerobic growth condition. Relative expression of the Eg-PNO gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 16 is a bar graph of the relative gene expression of the chloroplast gene, Chl-ccsA under heterotrophic & aerobic growth condition. Relative expression of the Chl-ccsA gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error. [0036] FIG.
  • 17 is a bar graph of the relative gene expression of the first carotene biosynthesis gene, Eg-crtB under mixotrophic & aerobic growth condition. Relative expression of the Eg-crtB gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 18 is a bar graph of the relative gene expression of the cell division controlling gene, Eg-CycA ( Cyclin A) under mixotrophic & aerobic growth condition. Relative expression of the Eg-CycA ( Cyclin A) gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 19 is a bar graph of the relative gene expression of the cell division controlling gene, Eg-CDK-A ( Cyclin Dependent Kinase A) under mixotrophic & aerobic growth condition. Relative expression of the Eg-CDK-A gene for WT, B2, and B3 cell lines over several culture time points. Error bars represent standard error.
  • FIG. 20 is a bar graph of the relative gene expression levels of the 2 paramylon synthase genes ( Eg-GSLl , Eg-GSL2 ), the 3 paramylon degradation genes ( Egcell 7 A, EgcelSl A, E gee 181 B), in comparison with the fatty acid biosynthesis first step gene, Trans-2-enoyl-coA reductase ( Eg-TER ). Error bars represent standard error.
  • FIG. 21A is a PCR gel image of a selection of homozygous CRISPR in frame knock-in cell lines, and it is a representation of successful knock-in of the hygromycin resistance gene - Out of the six CRISPR in- frame knock-in single colony cell lines, only one homozygous CRISPR in-frame knock-in cell line, SC2, carrying the hygromycin phosphotransferase gene (HPTII/HygR) was obtained.
  • the ssODN-HygR represents the single-stranded oligodeoxynucleotides of the hygromycin resistance gene donor template used as a negative control for the detection of the CRISPR in-frame knock-in sites that are beyond the ssODN-HygR donor template; NTC no DNA template control, WT wild type.
  • FIG. 2 IB is a representation of successful knock-in of the mGFP gene and the SARS-CoV-2 Spike Protein Subunit 1 (SP-S1) domain into endogenous regulatory elements (arrow represents the expected size of mGFP and SP-S1).
  • FIG. 22 are representations of hygromycin phosphotransferase (Hptll) protein expression after successful knock-in of the hygromycin resistance (HygR) gene in Euglena gracilis (left: SDS-page gel; right: immunoblot).
  • Hptll hygromycin phosphotransferase
  • the present disclosure relates to a microalgae organism with alterations in a microalgae gene (e.g., non-transgenic or transgenic human-induced alterations), a method of altering a trait in a microalgae organism, a method of expressing a heterologous nucleic acid molecule in a target genome of a microalgae organism, a composition for expressing a heterologous nucleic acid molecule in a target genome site of a microalgae organism, a method of expressing heterologous nucleotide molecules in a Euglena gracilis strain Z, line B2 and B3 organism, and methods of producing proteins, lipids, amino acids, and/or fatty acids with a microalgae organism.
  • a microalgae gene e.g., non-transgenic or transgenic human-induced alterations
  • 5 g, 6 g, and 7 g are also explicitly disclosed, as well as the range of values greater than or equal to 1 g and the range of values less than or equal to 8 g.
  • “Complementarity,” as used herein, refers to nucleic acid structure where sequences form double-stranded structures when matching, or complementary base pairs are present.
  • a sequence can be complementary to genomic DNA.
  • a sequence can be complementary to homologous arms present in nucleic acid molecule.
  • “Dry weight” means weight determined in the relative absence of water.
  • reference to a dry mixture refers to a specified percentage of a particular component(s) by dry weight as a percentage and is calculated based on the weight of the composition before any liquid has been added.
  • fatty acid refers to carboxylic acid consisting of a hydrocarbon chain and a terminal carboxyl group.
  • saturated fatty acid refers to a carboxylic acid with a hydrocarbon chain comprised of single bonds between hydrocarbons.
  • unsaturated fatty acid refers to a carboxylic acid with a hydrocarbon chain with one or more double bonds between hydrocarbons.
  • long chain fatty acid refers to a carboxylic acid with 14 or more carbons.
  • heterotroph refers to an organism, such as a microorganism including Euglena, such that it obtains nutrients substantially entirely from exogenous sources of organic carbon, such as carbohydrates, lipids, alcohols, carboxylic acids, sugar alcohols, proteins, or combinations thereof.
  • Euglena is a heterotroph where it is grown and propagated in conditions where there is substantially no light and an external carbon source.
  • Heterotrophic conditions or cultivation means cells are grown and propagated with an external carbon source and with substantially no light.
  • mixtureotroph refers to an organism, such as a microorganism including Euglena, that can obtain nutrients by both (1) photon capture to acquire energy (phototrophic or autotrophic), and (2) exogenous sources of organic carbon, such as carbohydrates, lipids, alcohols, carboxylic acids, sugar alcohols, proteins, or combinations thereof (heterotrophic).
  • Mixotrophic conditions or cultivation means cells are grown and propagated with light and an external carbon source.
  • microalgae refers to photosynthetic organisms of multiple phylogenetic groups and includes numerous unicellular and multicellular species.
  • microalgae as used herein includes organisms of the following phylogenetic groups: Chlorophyta (green algae, which includes mostly fresh water species); Phaeophyta (brown algae, which includes mostly marine species); Rhodophyta (red algae, which includes mostly marine species); Chrysophyta; Xanthophyta; Bacillariophyta; Euglenophyta (e.g., Euglena sp., Lepocinclis sp., Phacus sp., Trachelomonas sp., Asti asp., Colacium sp., Peranema sp., Petalomonas sp.);
  • microalgae can lack chloroplasts, have under developed chloroplasts, or have biologically non-functional chloroplasts. In some embodiments, the microalgae have chloroplasts.
  • phototroph or “autotroph” or derivatives thereof, as used herein, refers to an organism, such as a microorganism including Euglena, that it can carry out photon capture to acquire energy. For example, when an organism is phototrophic, it carries out photosynthesis to produce energy. Phototrophic or autotrophic conditions means cells are grown and propagated in the presence of light.
  • transgenic As used herein, the terms “transgenic,” “genetically modified organism,” or “GMO” are used interchangeably to indicate an organism in which foreign DNA from an unrelated organism has been artificially introduced.
  • a “non-transgenic organism” or “non-genetically modified organism” (“non-GMO”) comprises altered microorganisms in which no foreign DNA has been introduced or in which no foreign DNA remains in the microorganism.
  • a non-transgenic microorganism or non-GMO microorganism has DNA removed as compared to a wild-type microorganism
  • the present disclosure relates to microorganisms with human-induced alterations in their genome, e.g., having additional heterologous nucleic acid molecules added to their genome (including chloroplast, mitochondrial DNA) or having portions of genomic DNA (including chloroplast, mitochondrial DNA) removed [0055]
  • One aspect of the present disclosure relates to a Euglena organism comprising a non-transgenic, human-induced alteration in the GSL2 gene.
  • a Euglena organism comprising a transgenic, human-induced alteration in the GSL2 gene.
  • a Euglena organism can also have a property selected from the group consisting of (i) reduced carbohydrate content, (ii) reduced size of paramylon granules, (iii) increased protein content, (iv) increased lipid content, (v) altered amino acid profile, (vi) altered fatty acid profile, and (vii) any combination thereof compared to a wild type Euglena organism of a same species comprising a wild type GSL2 gene.
  • Paramylon is an abundant ⁇ - 1 ,3-glucan carbohydrate in Euglena.
  • the mRNA nucleic acid molecule for the ⁇ - 1 ,3-glucan synthase-like 2 gene ( GSL2 ) from Euglena gracilis is available in GenBank as accession number LC225615, which is hereby incorporated by reference in its entirety.
  • the nucleic acid molecule for GSL2 coding region is SEQ ID NO: 1.
  • the protein translation of the nucleic acid molecule for GSL2 coding region (SEQ ID NO: 1) is also available in GenBank as accession number BAX37083, which is hereby incorporated by reference in its entirety.
  • the GSL2 protein is shown in amino acid sequence is SEQ ID NO:2.
  • the alignment compares the partial genomic sequence of the wild type check line (WT-gEg-GSL2, SEQ ID NO:3), the partial genomic sequence of the B2 and B3 lines (B2/3-gEg-GSL2, SEQ ID NO:5), the coding sequence of the wild type check line (WT-gEg-GSL2_CDS, SEQ ID NO:4), and the coding sequence of the B2 and B3 lines (B2/3-gEg-GSL2_CDS, SEQ ID NO:6) showing the location of the 6 bp nucleotide deletion in the GSL2 gene of B2 cells compared with both the genomic and coding sequences in the wild type Euglena gracilis strain Z GSL2 gene.
  • the location of the protospacer adjacent motif (“PAM”) next to the target sequence of the guide RNA (GGG) is indicated with bold font.
  • the coding region of GSL2 aligned with and is identical with the genomic DNA is indicated with asterisks (SEQ ID NOs:3,4,5,6).
  • the alignment compares the partial genomic sequence of the wild type check line (WT-gEg-GSL2, SEQ ID NO:3), the partial genomic sequence of the B2 and B3 lines (B2/3-gEg-GSL2, SEQ ID NO:5), the coding sequence of the wild type check line (WT-gEg-GSL2_CDS, SEQ ID NO:4), and the coding sequence of the B2 and B3 lines (B2/3-gEg-GSL2_CDS, SEQ ID NO:6) showing the location of the 6 bp nucleotide deletion in the GSL2 gene of B2 cells compared with both the genomic and coding sequences in the wild type Euglena gracilis strain Z GSL2 gene.
  • the location of the protospacer adjacent motif (“PAM”) next to the target sequence of the guide RNA (GGG) is indicated with bold font.
  • the coding region of GSL2 is indicated with asterisks.
  • Splice junctions at the beginning and ends of introns are indicated in bold font and ## symbols.
  • the microalgae organism is a Euglena organism having an altered GSL2 gene such that the Euglena organism does not express a GSL2 protein or expresses a biologically non-functional GSL2 protein.
  • a Euglena organism has a human-induced, non-transgenic, alteration in its GSL2 gene.
  • An example of an alteration of a GSL2 gene in the partial genomic sequence of a GSL2 gene from Euglena gracilis strain Z to create new Euglena lines B2 and B3 is shown in SEQ ID NO:5.
  • SEQ ID NO:6 A partial nucleic acid molecule for the genomic region of SEQ ID NO:5 is shown SEQ ID NO:6 demonstrates 2 amino acids (methionine at position 56 (M56) and glycine at position 57 (G57)) deleted from GSL2
  • the microalgae organism of the present disclosure is a microalgae of the genus Euglena having an alteration in its GSL2 gene, and that alteration is homozygous, or occurs in two identical alleles.
  • the microalgae organism of the present disclosure is a microalgae of the genus Euglena and that Euglena organism does not express biologically functional paramylon.
  • alteration in the GSL2 gene of a Euglena organism is a deletion of about 10 base-pairs or less.
  • the alteration in the GSL2 gene is a deletion of 10 base-pairs, 9 base-pairs, 8 base-pairs, 7 base-pairs, 6 base-pairs, 5 base-pairs, 4 base-pairs, 3 base-pairs, 2 base-pairs, or 1 base-pair.
  • the entire GLS2 gene is deleted. In other embodiments more than 20, 50, 100, 200, 300, 400, 500, 1,000, 1,500, 2,000 or more base pairs are deleted. Any deletion that results in a non-biologically active GLS2 protein can be used.
  • an alteration in the GSL2 gene of a Euglena organism is an in-frame deletion.
  • An in-frame deletion involves 3 base-pairs, or multiples of 3 base-pairs resulting in the deletion of an amino acid (or more than one amino acid) in a protein.
  • the in-frame deletion is 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33 base-pairs.
  • the alteration in the GSL2 gene is a 6 base-pair deletion.
  • the Euglena organism with an altered GSL2 gene comprises underdeveloped chloroplasts or no chloroplasts compared to a wild type Euglena organism of a same species.
  • the Euglena organism does not comprise a chloroplast genome.
  • the Euglena organism optionally does not comprise a chloroplast genome.
  • the Euglena is optionally resistant to hygromycin or other antibiotic. Resistance to hygromycin can be due to the insertion of a hygromycin resistance gene (e.g. HPTII encoding for hygromycin phosphotransferase) into the Euglena genome.
  • a hygromycin resistance gene e.g. HPTII encoding for hygromycin phosphotransferase
  • the cell line B2 does not comprise chloroplasts.
  • the presence or absence of chloroplasts can be inherited differently due to asymmetric cell divisions between cell lines.
  • a Euglena chloroplast genome is deleted or one or more genes of the chloroplast genome are deleted such that chloroplasts are not present, are non-functional, or are underdeveloped.
  • the alterations to the chloroplast genome can be produced using any methodology known in the art, including, for example using CRISPR technologies.
  • the Euglena organism with an altered GSL2 gene comprises a trait associated with the altered GSL2 gene, which trait does not appear in a Euglena of the same species.
  • the trait is a property selected from the group consisting of (i) reduced carbohydrate content, (ii) reduced size of paramylon granules, (iii) increased protein content, (iv) increased lipid content, (v) altered amino acid profile, (vi) altered fatty acid profile, and (vii) any combination thereof compared to a wild type Euglena organism of a same species.
  • the Euglena organism comprises one or more of the above properties when grown under heterotrophic aerobic conditions or heterotrophic anaerobic conditions.
  • the Euglena organism with an altered GSL2 gene comprises one or more of the above properties when grown under heterotrophic aerobic conditions.
  • the Euglena organism with an altered GSL2 gene comprises the property when grown under heterotrophic anaerobic conditions.
  • a Euglena organism with an altered GSL2 gene comprises the property when grown under mixotrophic anaerobic conditions, mixotrophic aerobic conditions, phototrophic anaerobic conditions, phototrophic aerobic conditions, heterotrophic aerobic conditions, or heterotrophic anaerobic conditions.
  • a Euglena organism with an altered GSL2 gene comprises a reduced amount of carbohydrate content compared to a wild type Euglena of the same species.
  • a Euglena organism with an altered GSL2 gene comprises less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4% less than about 3%, less than about 2%, or less than about 1% carbohydrate content.
  • the Euglena organism with an altered GSL2 gene comprises reduced carbohydrate contents of less than about 5%. In another particular embodiment, a Euglena organism with an altered GSL2 gene comprises reduced carbohydrate contents of less than about 3%. In one embodiment the carbohydrate content is calculated by a dry weight basis of biomass (“dry weight”). In another embodiment the carbohydrate content is calculated on an “as is” biomass weight, which includes moisture. In an embodiment a Euglena organism with an altered GSL2 gene has about 1, 5, 10, 20, 30, 40, 50, 60, 70% or less carbohydrate content as compared to a wild-type Euglena organism.
  • GSL2 gene are not detectable in the organism using microscopy.
  • An insoluble, linear ( 1 ,3)-[3-glucan of high molecular mass, paramylon occurs naturally in a high crystalline form in discrete membrane-bound granules in the cytoplasm of euglenid protozoans ( e.g ., Euglena gracilis) (Bruce A. Stone, “Chemistry of b-Glucans,” in Chemistry,
  • paramylon granules of an altered Euglena organism have a smaller or reduced size compared to paramylon granules of a wild type Euglena organism (/. e. , a Euglena organism without an altered genome or GSL2 gene).
  • a Euglena organism with an altered GSL2 gene has about paramylon granules that are about 1, 5, 10, 20, 30, 40, 50, 60, 70% or more smaller paramylon granules of a wild-type Euglena organism.
  • an altered Euglena organism produces no paramylon granules.
  • the Euglena organism with an altered GSL2 gene comprises an increased or elevated protein content compared to a Euglena of the same species.
  • the Euglena organism with an altered GSL2 gene comprises a protein content of at least about 60%.
  • the Euglena organism with an altered GSL2 gene comprises an increased or elevated protein of at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, or at least about 70%.
  • the Euglena organism with an altered genome comprises a protein content of at least about 60%.
  • the Euglena organism with an altered genome comprises a protein content of at least about 65%.
  • protein content is calculated on a dry weight biomass basis (dry weight).
  • protein content is calculated on an “as is” biomass weight basis, which includes moisture.
  • the protein content of the Euglena organism with an altered genome is compared to the protein content of a wild type Euglena ( i.e ., a Euglena organism with an altered genome).
  • a Euglena organism with an altered GSL2 gene has about 1, 5, 10, 20, 30, 40, 50, 60, 70% or more protein content as compared to a wild-type Euglena organism.
  • the Euglena organism with an altered GSL2 gene comprises an altered amino acid profile compared to a wild type Euglena of the same species.
  • the Euglena organism with an altered genome comprises an increased amount or occurrence of one or more amino acids such as aspartic acid, glutamic acid, serine, histidine, glycine, threonine, tyrosine, methionine, phenylalanine, leucine, lysine, or total amino acids, or any combination thereof as compared to a wild type Euglena organism of a same species.
  • a Euglena organism with an altered GSL2 gene has about 1, 5, 10, 20, 30, 40, 50, 60, 70% or more total amino acid content or about 1, 5, 10, 20, 30, 40, 50, 60, 70% or more or one or more of aspartic acid, glutamic acid, serine, histidine, glycine, threonine, tyrosine, methionine, phenylalanine, leucine, lysine, or total amino acids, or any combination thereof as compared to a wild type Euglena organism of a same species as compared to a wild-type Euglena organism.
  • the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid aspartic acid.
  • the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid glutamic acid. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid serine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid histidine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid glycine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid threonine. In some embodiments, the Euglena organism with an altered genome has increased amount or occurrence of the amino acid tyrosine.
  • the Euglena organism with an altered genome has increased amount or occurrence of the amino acid methionine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid phenylalanine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid leucine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of the amino acid lysine. In some embodiments, the Euglena organism with an altered genome has an increased amount or occurrence of total amino acids. In some embodiments, the amino acid level of the Euglena organism with an altered genome is compared to the level of the amino acid in a wild type Euglena organism of the same species.
  • the Euglena organism with an altered GSL2 gene has an elevated lipid content compared to a wild type Euglena of the same species.
  • the Euglena organism with an altered genome may have an increased lipid content of greater than about 12%, greater than about 13%, greater than about 14%, greater than about 15%, greater than about 16%, greater than about 17%, greater than about 18%, greater than about 19%, or greater than about 20%.
  • the Euglena organism with an altered genome comprises an increased lipid content of greater than about 15%.
  • a lipid may be a fat or an oil.
  • lipid content is calculated on a dry weight biomass basis (dry weight).
  • the lipid content is calculated on an “as is” biomass weight basis, which includes moisture.
  • the lipid content of the Euglena organism with an altered genome of the present disclosure is compared to the lipid content of a wild type Euglena, or a Euglena without the altered genome.
  • a Euglena organism with an altered GSL2 gene has about 1, 5, 10, 20, 30, 40, 50, 60, 70% or more lipid content as compared to a wild-type Euglena organism.
  • a Euglena organism with an elevated lipid content comprises an increased amount or occurrence of one or more of myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, or any combination thereof, compared to a Euglena organism of a same species without an altered genome.
  • the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid myristic acid.
  • the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid palmitic acid.
  • the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid stearic acid.
  • the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid oleic acid. In some embodiments, the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid linolenic acid. In some embodiments, the Euglena organism of the present disclosure has an increased amount or occurrence of the fatty acid myristic acid. In some embodiments the fatty acid content of the organism of the present disclosure is calculated on a dry weight biomass basis (dry weight). In some embodiments the fatty acid content of the organism of the present disclosure is calculated on an “as is” biomass weight basis, which includes moisture.
  • the fatty acid level (e.g ., amount or occurrence) of the organism of the present disclosure is compared to the fatty acid level in a wild type Euglena organism of a same species.
  • a Euglena organism with an altered GSL2 gene has about 1, 5, 10, 20, 30,
  • the microalgae is a Euglena organism selected from any species of the genus Euglena. Such species include, without limitation,
  • the Euglena organism is Euglena gracilis strain
  • the Euglena organism is Euglena gracilis strain Z, line B2.
  • the Euglena organism is Euglena gracilis strain Z, line B3. [0077] In some embodiments, the organism is a microalgae of the genus
  • the organism may be a species of the genus Chlorella or Schizochytrium selected from Chlorella autotrophica, Chlorella colonials, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis, Chlorella vulgaris, Schizochytrium aggregatum, Schizochytrium limacinum, and Schizochytrium minutum.
  • a microorganism is Euglena sp., Lepocinclis sp.,
  • Another aspect of the present disclosure relates to a method of altering a trait in a Euglena organism.
  • This method involves providing a ribonuclear protein (RNP) complex comprising a guide RNA to an endogenous gene of a Euglena organism and a clustered regularly interspaced short palindromic repeat (CRISPR) Cas-related nuclease.
  • the RNP complex is introduced into a Euglena organism to affect a genetic alteration.
  • the method can optionally further involves selecting for the genetic alteration in the organism, where the organism comprises the genetic alteration and a property selected from the group consisting of (i) reduced carbohydrate content, (ii) increased protein content, (iii) increased lipid content, (iv) altered amino acid profile, (v) altered fatty acid profile, (vi) reduced or underdeveloped chloroplasts, and (vii) any combination thereof compared to a wild type Euglena organism of a same species.
  • the genetic alteration is homozygous. In some embodiments, the genetic alteration is in the GSL2 gene, but any gene or non-coding sequence can be targeted using CRISPR technologies.
  • ribonucleoproteins (“RNPs”) and CRISPR may be designed to target a specific nucleic acid sequence in a Euglena cell for gene editing to create the altered Euglena of the present disclosure. Methods for making alterations using RNPs and CRISPR are described in detail infra.
  • RNPs and CRISPR may be used to modify the genome of a Euglena organism without introducing foreign DNA into the genome. The use of RNPs and CRISPR to modify a gene is considered a non-transgenic approach, since foreign DNA is not introduced into the organism.
  • RNP structures capable of modifying the genome of an organism are man made structures comprising a guide RNA (“gRNA”) and an RNA-guided endonuclease, or Cas-related nuclease.
  • RNA- guided endonucleases are from a CRISPR/Cas system, which can be a type I, a type II, or a type III system. Use of such systems for gene editing has been described.
  • a guide RNA is designed based on the sequence of the gene targeted for modification in an organism’s ( e.g ., a microalgae of the genus Euglena ) genome.
  • the gRNA is made up of two parts: crispr RNA (“crRNA”), a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (“tracrRNA”), which serves as a binding scaffold for the Cas-related nuclease.
  • crRNA crispr RNA
  • tracrRNA trans-activating crRNA
  • the guide RNA can be one molecule that combines the crRNA and tracrRNA.
  • the Cas-related nuclease is Cas9, but can also be an endonuclease from one of many related CRISPR systems that have been described.
  • Non limiting examples of Cas-related nucleases include Casl, CaslB, Cas2, Cas3, Cas4,
  • Cas5, Cas6, Cas7, Cas8, Cas9 also known as Csnl and Csxl2
  • CaslO Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX,
  • the gRNA/Cas-related nuclease complex is recruited to a target sequence by the base-pairing between the gRNA sequence which has a region of complementarity to the target sequence in the DNA of Euglena.
  • the genomic target sequence must also contain the correct Protospacer Adjacent Motif (“PAM”) sequence immediately following the target sequence.
  • PAM Protospacer Adjacent Motif
  • the binding of the gRNA/Cas complex localizes the Cas-related nuclease to the genomic target sequence so that the Cas-related nuclease can cut both strands of DNA causing a Double Strand Break (“DSB”).
  • DSB Double Strand Break
  • a DSB can be repaired through one of two general repair pathways: (1) the non-homologous end joining (“NHEJ”) DNA repair pathway or (2) the homologous directed repair (“HDR”) pathway.
  • NHEJ repair pathway often results in insertions/deletions (“indels”) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (“ORF”) of the targeted gene.
  • the HDR pathway requires the presence of a repair template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence.
  • Specific nucleotide changes and insertions can be introduced into a targeted gene by the use of HDR with a repair template such as a single stranded oligo donor nucleotide (“ssODN”).
  • CRISPR specificity can be controlled by level of homology and binding strength of the specific gRNA for a given gene target, or by modification of the Cas endonuclease itself. For example, a D10A mutant of the RuvC domain retains only the HNH domain and generates a DNA nick rather than a DSB.
  • the methods or CRISPR/Cas gene editing can be used to generate a gene knockout in a microalgae organism (e.g ., a microalgae of the genus Euglena ) by causing small nucleotide insertions or deletions (indels) at the DSB site.
  • a Euglena organism gene can be activated or repressed by modifying transcription of target genes or pathways in regulatory regions, such as promoters or transcription start sites.
  • a point mutation or multiple point mutations can be generated in a polynucleotide of interest in the genome of a microalgae of the genus Euglena.
  • multiple gene targets can be modified using CRISPR/Cas in a single experiment, where such gene targets can be homologs targeted by identical or related guide RNAs, or may be unrelated genes separately targeted using multiple guide RNAs having specificity for the different gene targets.
  • CRISPR/Cas gene editing can be attained.
  • a program can be used to provide a prediction of the ability of a guide RNA to edit a target gene.
  • a program is described in Doench et al., “Rational Design of Highly Active sgRNAs for CRISPR-Cas9 Mediated Gene Inactivation,” Nat. Biotechnol. 32:1262-1267 (2014), which is hereby incorporated by reference in its entirety, which establishes a “Doench score” for making a prediction of a gRNA’s ability to edit a target gene.
  • the guide RNA has a Doench score greater than about 0.4.
  • a guide RNA targets e.g., SEQ ID NO:7 (described infra), but any microalgae DNA can be targeted.
  • DNA fragments, and the like into a microalgae can be accomplished using a variety of transformation techniques.
  • transformation is achieved by infection with a microbe, such as Rhizobia or Agrobacterium, electroporation, polyethylene glycol (“PEG”)-mediated DNA transfer, microinjection, particle bombardment, or vacuum infiltration.
  • PEG polyethylene glycol
  • the RNP complex is introduced into the microalgae cells using electroporation.
  • a further aspect of the present disclosure relates to a method of expressing a heterologous nucleic acid molecule in a target genome insertion site of a microalgae ( e.g ., of the genus Euglena ) organism.
  • This method involves providing an RNP complex comprising a guide RNA having complementarity to an endogenous gene of a microalgae organism and a CRISPR Cas-related nuclease.
  • the method further involves providing an ssODN comprising a first homology arm, a heterologous nucleic acid molecule, and a second homology arm, where the first and second homology arms have complementarity to regions flanking the target genome insertion site.
  • This method further involves introducing the RNP complex and the ssODN into a microalgae organism, where the heterologous nucleic acid molecule is inserted into the target genome insertion site and is expressed.
  • heterologous nucleic acid molecules incorporated into a microorganism can provide for, e.g., expression of foreign proteins in the microorganism.
  • Heterologous nucleic acid molecules can also refer to nucleic acid molecules native to the organism but positioned in a heterologous location in the genome compared to their native location.
  • heterologous nucleic acid molecules can provide for the overexpression of proteins native to a microalgae species or organism, or for reduced expression of native microalgae genes.
  • a heterologous nucleic acid molecule inserted into a microalgae genome provides for expression of a foreign protein or the overexpression of a native or altered microalgae protein.
  • Such embodiments provide a microalgae organism having an improved trait or traits as the result of expression of the heterologous nucleic acid molecule encoding a polypeptide or polypeptides of interest.
  • a heterologous nucleic acid molecule to be inserted into the microalgae can be greater than about 100 nucleotide base pairs (bp). In some embodiments, the heterologous nucleic acid molecule can be about 100 bp to about 3000 bp. In some embodiments, the heterologous nucleic acid molecule can be greater than about 500 bp. In some embodiment, the heterologous nucleic acid molecule can be greater than about 1000 bp. In some embodiments, the heterologous nucleic acid molecule can be greater than about 1 bp, 10 bp, 50 bp, 100 bp. In some embodiments, the heterologous nucleic acid molecule can be greater than about 1500 bp, 2,000 bp, 2,500 bp, 3,000 bp, 3,500 bp, 4,000 bp or more.
  • a single strand oligo donor nucleotide has sequences complementary to the DSB ends generated by cleavage in the genome by the genome editing nuclease, called a homology arm for precise in-frame CRISPR knock-in of a gene of interest.
  • the term “knock-in” as used herein encompasses both insertion of a donor sequence such as a heterologous nucleic acid molecule for a gene of interest into a genome (including to chloroplast, mitochondrial, and plasmid DNA), and replacement of a sequence in a genome by a donor sequence.
  • target site refers to a nucleotide sequence in the genome (including chloroplast, mitochondrial DNA, plasmid DNA) of a cell at which a single or double-strand break is induced in the cell genome by a Cas-related endonuclease.
  • the target site can be an endogenous site in the genome of a cell.
  • the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell.
  • a target site can be found in a heterologous genomic location (including to chloroplast, mitochondrial, and plasmid DNA) compared to where it occurs in nature.
  • a heterologous nucleic acid molecule is in frame at the endogenous genome insertion site. This means that the heterologous nucleic acid molecule is inserted such that it has the same reading frame as the endogenous gene.
  • a promoter refers to a nucleic acid molecule capable of controlling transcription of another nucleic acid molecule.
  • a promoter is a non-coding genomic DNA sequence, usually upstream (5') to the relevant nucleic acid molecule, and its primary function is to act as a binding site for RNA polymerase to initiate transcription by the RNA polymerase.
  • Promoter sequences in general include proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • a promoter is capable of controlling expression, capable of initiating transcription, or driving expression of a DNA sequence when it is able to carry out this primary function of a promoter.
  • Promoter function includes expression of RNA, including functional RNA, or the expression of a polypeptide for operably linked encoding nucleotide sequences, as the transcribed RNA ultimately may be translated into the corresponding polypeptide. Promoters vary in their strength (i.e., their ability to promote transcription). The nucleotide sequence of the promoter determines the nature of the RNA polymerase binding and other related protein factors that attach to the RNA polymerase and/or promoter, and the rate of RNA synthesis. In some embodiments the expression of a heterologous nucleic acid molecule is driven by the GSL2 promoter. In some embodiments expression of a heterologous nucleic acid molecule is driven by a Egcell 7 A promoter.
  • the GSL2 promoter or Egcell 7 A promoter are operably linked to the heterologous nucleic acid molecule.
  • the genomic sequence upstream of the start codon of the GSL2 gene that includes the promoter and 5' untranslated region (“5'UTR” indicated in bold) is shown in SEQ ID NO: 8.
  • the ssODN also comprises a donor sequence to be inserted into the genome at the DSB.
  • the sequence to be inserted is a heterologous nucleic acid molecule.
  • the ssODN also has a second homology arm, with a first and a second homology arm on each end of the donor DNA.
  • the length of each homology arm is about 10 to about 100 bases, about 12 to about 80 bases, or about 15 to about 60 bases.
  • the first homology arm is complementary to GSL2.
  • the first homology arm is SEQ ID NO:9.
  • the first homology arm is complementary to Egcell 7 A.
  • the first homology arm is SEQ ID NO:73.
  • the second homology arm is complementary to GSL2.
  • the second homology arm is SEQ ID NO: 10.
  • the second homology arm is complementary to EgcelHA.
  • the second homology arm is SEQ ID NO:74.
  • the sequence to be inserted is a coding region of a gene of interest.
  • the heterologous nucleic acid molecule is mGFP or the SARS-COV-2 spike protein subunit 1.
  • the heterologous nucleic acid molecule is GFP (SEQ ID NO: 11) or the SARS-COV-2 spike protein subunit 1 (SEQ ID NO: 12).
  • the heterologous nucleic acid molecule is hygromycin resistance (HygR) gene (SEQ ID NO: 129).
  • the guide RNA has complementarity to the GSL2 gene.
  • the guide RNA targets a region of the GSL2 gene (SEQ ID NO: 13).
  • the ssODN has homology arms to GSL2 and a nucleic acid molecule for GFP (SEQ ID NO: 14) or homology arms to GSL2 and a nucleic acid molecule for the SARS-COV-2 spike protein subunit 1 (SEQ ID NO: 15).
  • the guide RNA is to the Egcell 7 A gene. In some embodiments, the guide RNA has complementarity to and targets a region of the Egcell 7 A gene.
  • the ssODN has homology arms to Egcell 7 A and a nucleic acid molecule for mGFP (SEQ ID NO: 84) or homology arms to Egcell 7 A and a nucleic acid molecule for the SARS-COV-2 spike protein subunit 1 (SEQ ID NO:81) or homology arms to Egcell 7 A and a nucleic acid molecule for hygromycin resistance gene (SEQ ID NO:68).
  • genome modifications include, without limitation, modification of paramylon, carbohydrate, protein, lipid, and/or other metabolic pathways to provide microorganisms having increased or decreased levels of specific compounds including industrially useful compounds, and/or enabling production of pharmaceutically relevant compounds.
  • genome modifications include to chloroplast, mitochondrial, and plasmid DNA
  • the heterologous nucleic acid molecule of interest is one or more of a reporter gene nucleic acid molecule, a transcriptional regulator nucleic acid molecule, a viral nucleic acid molecule, a bacterial nucleic acid molecule, a eukaryotic nucleic acid molecule, a yield enhancing nucleic acid molecule, a disease resistance nucleic acid molecule, a nutritional quality nucleic acid molecule, a pharmaceutical nucleic acid molecule, a selectable marker nucleic acid molecule, and a synthetic nucleic acid molecule.
  • An additional aspect of the present disclosure relates to a composition for expressing a heterologous nucleic acid molecule in a target genome insertion site of a microalgae ( e.g . of the genus Euglena ) organism.
  • the composition comprises an ssODN, where the ssODN comprises a first homology arm, a nucleotide sequence encoding a heterologous nucleic acid molecule, and a second homology arm, where the first and second homology arms have complementarity to regions flanking the target genome insertion site (e.g., the region in the microalgae where the heterologous nucleic acid molecule will be inserted).
  • the composition also includes a guide RNA having complementarity to an endogenous gene of a microalgae organism and a CRISPR Cas- related nuclease, where the Cas-related protein cleaves the target genome insertion site and where the sequence encoding the heterologous nucleic acid molecule is inserted into the target genome insertion site.
  • Another aspect of the present disclosure relates to a method of expressing a heterologous nucleic acid molecule in a Euglena gracilis strain Z, line B2 organism.
  • This method involves transforming a Euglena gracilis strain Z, line B2 organism with a DNA vector comprising a heterologous nucleic acid molecule, a promoter operably linked to the heterologous nucleic acid molecule, and a selectable marker, where said transforming expresses the nucleic acid molecule in the organism.
  • Selectable markers can be used to select for cells that comprise a DNA construct. Selection of transformed cells comprising the DNA construct utilizes an antibiotic or other compound useful for selective growth as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present in the vector with which the host cell was transformed.
  • the marker may encode biocide resistance, antibiotic resistance (e.g ., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.).
  • selectable markers include, but are not limited to, a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant DHFR gene.
  • a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.
  • a bar gene which codes for bialaphos resistance
  • a mutant EPSP synthase gene which encodes glyphosate resistance
  • a nitrilase gene which confers resistance to bromoxynil
  • ALS acetolactate synthase gene
  • selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin, tetracycline, etc. Examples of selectable markers are described in, e.g., U.S. Patent Nos. 5,550,318; 5,633,435; 5,780,708; and 6,118,047, which are each hereby incorporated by reference in their entirety.
  • the selectable marker is selected from the group consisting of genes conferring resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline.
  • the selectable marker is for hygromycin resistance.
  • the selectable marker is capable of selecting for the vector at about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 pg/ml hygromycin or any intervening concentration.
  • the selectable marker is capable of selecting for the vector at about 30 pg/ml hygromycin.
  • This aspect of the present disclosure can be carried out according to any of the embodiments disclosed herein.
  • Another aspect of the present disclosure relates to a method of producing proteins, lipids, amino acids, fatty acids, or combinations thereof. This method involves culturing a microalgae organism that does not express a biologically functional glucan synthase-like 2 (GSL2) protein in a medium and harvesting the proteins, lipids, amino acids, fatty acids, or combinations thereof.
  • GSL2 biologically functional glucan synthase-like 2
  • the microalgae organism is non-transgenic.
  • the fatty acids are saturated fatty acids, long chain unsaturated fatty acids, or combinations thereof.
  • the protein, lipid, amino acids, fatty acids, or combinations thereof are expressed by the Euglena organism from one or more heterologous nucleic acid molecules.
  • the microalgae organism expresses a heterologous protein that is harvested.
  • the microalgae organism is cultured in the presence of light. In some embodiments, the microalgae organism is cultured in the absence of light. In some embodiments, the microalgae organism is cultured under mixotrophic anaerobic conditions, mixotrophic aerobic conditions, phototrophic anaerobic conditions, phototrophic aerobic conditions, heterotrophic aerobic conditions, or heterotrophic anaerobic conditions.
  • compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art.
  • the terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise.
  • compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
  • the following are provided for exemplification purposes only and are not intended to limit the scope of the embodiments described in broad terms above.
  • crRNA sequences were queried using the Doench CRISPR Target Activity Score calculation which predicts the ability of the guide RNA to knock out the target gene (Doench et al., “Rational Design of Highly Active sgRNAs for CRISPR-Cas9 Mediated Gene Inactivation,” Nat Biotechnol 32:1262-1267 (2014), which is hereby incorporated by reference in its entirety). Based on this analysis, a crRNA sequence with a Doench Activity Score of 0.427 was selected that was predicted to target a novel CRISPR site with a PAM sequence of GGG within the genomic sequence of Eg-GSL2 gene called Target 1-2: 5'-AGGATTTTCATCCGTAGCAT-3' (SEQ ID NO:7).
  • RNP-CRISPR/Cas9 complexes tested included RNP complexes to Target 1-2.
  • the wild type (WT) Euglena gracilis strain Z ( Euglena gracilis Klebs, ATCC 12894) cells were cultured under heterotrophic and aerobic conditions on an orbital shaker (120 rpm, 28°C) in glucose supplemented growth medium (pH 3.2) ( e.g . Hunter’s or Cramer-Meyer medium). After 3 days of growth, cells were collected by centrifugation at 2000 rpm and washed with an electroporation buffer (3:2 ratio of Growth medium pH 5.5 to 0.3M Sucrose). Cells were resuspended in the electroporation buffer to obtain a cell concentration of ⁇ 2 x 10 6 cells/mL.
  • QuantiTect Reverse Transcription Kit according to the manufacturer’s protocol and qRT- PCR was performed using the QuantStudioTM 3 Real-Time PCR System (Life Technologies, Carlsbad, CA).
  • the reaction volume was 20 pL, with 5 ng of cDNA, 1 pL of each gene-specific forward and reverse primers at 10 pM, and 10 pL of 2X PowerUp qRT-PCR SYBR Green Master Mix Buffer (including ROX as the internal passive fluorescent dye) (Qiagen Inc., Hilden, Germany).
  • the qRT-PCR thermo-cycling conditions include the following stages and steps: (i) Holding Stage: 50°C, 2 minutes; 95°C, 2 minutes; (ii) Cycling Stage: 40 cycles of 95°C for 3 seconds; 60°C for 30 seconds (Collect SYBR green fluorescence signal during the extension stage at this 60°C temperature); (iii) Melt Curve Stage: 95°C, 15 seconds; 60°C, 1 minutes; 95°C, 15 seconds (Collect SYBR green fluorescence signal during the dissociation stage from 60°C to 95°C).
  • the AACt was obtained based on the ACt value of a tested sample against the ACt value of a check (CK) at a certain time point, and the actin gene was used as endogenous control gene.
  • the relative gene expression ratio was calculated with the equation of 2 A -AACt.
  • Eg-GSL2 was identified as the functional paramylon biosynthesis gene with detectable expression levels, while Eg-GSLl had no detectable expression levels under the heterotrophic/aerobic growth conditions used (FIG. 1).
  • the primers used for gene expression evaluation are shown in Table 1.
  • Non-GMO genome edited Euglena gracilis lines were obtained by applying DNA-free ribonucleoprotein (RNP) complexes to edit the Eg-GSL2 gene without the introduction of foreign genetic material into the wild type Euglena gracilis strain Z cells.
  • RNP DNA-free ribonucleoprotein
  • No treatment control which is the wild type Euglena gracilis strain Z cells without any treatment and with genetically homozygous wild type alleles of the Euglena gracilis GSL2 ( Eg-GSL2-WT) gene was compared to the Target 1-2 CRISPR edited Euglena cells with an edit in Eg-GSL2 ( Eg-gsl2-mt ) at 72 hours after CRISPR treatment.
  • the large size and reduced number of paramylon granules exhibited were caused by the genetically heterozygous alleles of Eg-GSL2-WT/Eg-gsl2-mt.
  • Targetl-2 CRISPR edited single cell selected and cultured Euglena cells at 3 weeks after CRISPR treatment contained homozygous alleles of Eg-gsl2-mt/Eg-gsl2-mt.
  • Genome edited cell lines B2 and B3 were selected for further characterization to compare the difference between the B2 and B3 cell lines and their original wild type E. gracilis Z stain check (CK).
  • Eg- GSL2-cr ⁇ 5'-GTGGGGCATTGGAGGGTC-3' (SEQ ID O:44) and 73 ⁇ 4--G.SX2-crRl (5'- ACCATCCCATGACAGCTGG-3' (SEQ ID NO:45).
  • the ⁇ 650 bp genomic fragment was isolated and purified using the QIAquick PCR Purification Kit (Qiagen Inc., Hilden, Germany).
  • Eg-GSL2 genomic PCR fragments ( ⁇ 650 bp) holding the CRISPR target site from both sibling cell lines B2 and B3 were mixed with the wild type check (CK) PCR product, denatured at 95°C for 5 minutes, then cooled down to room temperature on lab bench to form the DNA duplex.
  • the duplex was cleaved using the T7E1 enzyme, which cleaves at mismatches between DNA formed between genome edited DNA and wild type DNA.
  • Lanes 1-3 had a fragment of DNA ( ⁇ 650 bp) amplified from the Eg- GSL2 gene containing the CRISPR Target 1-2 site.
  • Lane 1 had DNA amplified from line B2, lane 2 had DNA amplified from line B3, and lane 3 had DNA amplified from and unmodified check line (CK1).
  • Lane 4 had DNA amplified from the 18S gene marker as a positive control (CK2).
  • the ⁇ 650 bp fragment was cleaved into two parts, 420 bp and 232 bp due to the 6 bp deletion within the GSL2 gene in the B2 and B3 cell lines, indicating the presence of genome edits in these lines compared to the CK line DNA.
  • the unmodified check line (CK) was not cleaved by the T7E1 enzyme, whereas the mixture of CK+B2 DNA and CK+B3 DNA were cleaved by the T7E1 enzyme.
  • DNA size markers are in the lane marked “M”.
  • Eg-GSL2 genomic PCR fragments ( ⁇ 650 bp) holding the CRISPR target site from both sibling cell lines, B2 and B3, and the wild type check (CK) cells were sequenced at The Centre for Applied Genomics (TCAG, Toronto, Canada). An alignment of portions of the genomic sequence of the wild type check was shown in this region (SEQ ID NO:3), the genomic sequence of B2 and B3 (SEQ ID NO:5). This sequencing analysis showed that a 6 bp region was deleted from the Eg-GSL2 gene coding region in the genomic fragments holding the paramylon synthase CRISPR target sites in lines B2 and B3. B2 and B3 cell lines had the same deletion because they originated from a common single mother cell before separation and selection.
  • Eg-gsl2-mt This allele is called Eg-gsl2-mt to distinguish it from the wild type Eg-GSL2-WT allele.
  • the 6 bp deletion of Eg-gsl2-mt leaves the predicted translated protein in frame with the loss of two amino acids (M56 and G57).
  • the partial amino acid sequence of Eg-gsl2-mt (SEQ ID NO:46) is as follows:
  • Intron splice sites were identified with canonical AG/GT borders as well as non- canonical AG/AT, CC/TT, and CT/GA splice junction borders.
  • An allele-specific marker was developed to track and differentiate the Eg- gls2-mt allele in the B2 and B3 cell lines from the wild type and other Euglena gracilis strains.
  • the allele-specific TaqMan probes labeled with different fluorescent dyes were designed and synthesized together with a pair of Eg-GSL2 gene-specific primers for genotyping of B2, B3, and wild type Euglena gracilis cells. As shown in FIG.
  • the allelic discrimination assay showed that 3 replicates of B2 cells with 6 bp deletions ( Eg - gsl2-mt ) were grouped together (the top left comer in circles), 3 replicates of the wild type cells ( Eg-GSL2-WT) with no such deletion were grouped together (the bottom right comer, circles); while the negative controls were grouped together (the bottom left comer, squares).
  • Eg - gsl2-mt 3 replicates of B2 cells with 6 bp deletions
  • Eg-GSL2-WT wild type cells
  • Eg-GSL2 gene was used to obtain homozygous Eg-gsl2-mt/Eg-gsl2-mt cells. It was discovered that the homozygous RNP delivered CRISPR-Cas9 edited Eg-gsl2-mt/Eg- gsl2-mt cells of Euglena gracilis had no paramylon granules in the cells, and that after 3 weeks of cell culture, all the cells in the cell line showed the same phenotype. The homozygous Eg-gsl2-mt/Eg-gsl2-mt cells had similar viability, moving activity, and cell sizes as the original wild type Eg-GSL2-WT/Eg-GSL2-WT cells.
  • the new Euglena gracilis B2 and B3 sibling cell lines and their original wild type check (CK) strain Z were grown under the following growth and culture conditions: (i) mixotrophic and aerobic mixotrophic fermentation; (ii) mixotrophic and anaerobic mixotrophic fermentation; (iii) heterotrophic and aerobic fermentation; and (iv) heterotrophic and anaerobic fermentation.
  • the same type of glucose supplemented growth media pH 3.2
  • the cells were cultured in 1.0 liter of growth media in 2.0 liter of flasks in INFORS HT Multitron Incubators (INFORS-HT, Surrey, United Kingdom) with an internally installed full spectrum light panel at the light strength of 1600 pmol.
  • the incubation shakers were set at constant 28°C and 120 rpm.
  • the shakers were covered with aluminum sheets to shield the cultures from all light sources.
  • nitrogen gas (“N2”) was pumped into the flasks for 2 minutes, then cells were cultured for 48 hours.
  • the glucose content in the growing samples were measured daily. The measurements showed that there was a detectable difference in glucose consumption in B2 and B3 lines compared to the CK.
  • the cell cultures were continued until the glucose concentration went below 0.5 g/L.
  • the first set of samples to reach the concentration of ⁇ 0.5 gram of glucose/Liter was the wild type CK cells. Therefore, they were harvested on day 7, followed by B3 cells on day 8 and then B2 cells on day 9.
  • the glucose in the CK cell samples started to diminish starting at day 3 and declined faster than the B2 and B3 cell lines.
  • B2 and B3 cell lines consumed glucose at a significantly slower rate than the wild type but produced comparable amounts of fresh biomass compared to the wild type under heterotrophic conditions.
  • B2 cell numbers were measured at nine days after the glucose in the media was depleted ( ⁇ 0.5 g/L), reaching a concentration of 59.41 x 10 6 cells per milliliter.
  • B3 cells were measured after eight days when their glucose levels were below 0.5 g/L, reaching a concentration of 38.66 x 10 6 cells per milliliter CK cells were measured at seven days when their glucose levels were below 0.5 g/L, reaching a concentration of 24.62 x 10 6 cells per milliliter.
  • Many more cells were produced by the B2 and B3 lines using the same amount of carbon source (glucose) and other media components compared to the wild type CK.
  • the new cell line B3 a sibling of the B2 cell line, is genetically identical to B2 but inherently different from B2, because it still maintains the ability to make some levels of chlorophyll.
  • B2, B3, and WT cells were maintained under light treatment for over 7 days in 6-well plates.
  • For each cell line initially at day 0, two million cells were kept in 2 mL of growth media in each well. Two replicates were prepared for each cell for phenotypic observations. B2 cells never turned green, whereas B3 cells didn’t turn green until day 4. The wild type CK cells showed green color as early as at day 2 under light treatment.
  • the genome-specific gene markers used included a nuclear genome-specific marker gene: Oxygen evolving enhancer protein 1 , PsbO encoded by the nuclear gene nPsbO, two chloroplast genome-specific marker genes, PsbA (thylakoid membrane protein of Photosystem II) and rbcL (ribulose bisphosphate carboxylase large chain, RuBisCO large subunit for carbon dioxide fixation), and a mitochondrial genome- specific marker gene, COX1 (respiratory complex IV subunit 1 /Cytochrome c oxidase subunit 1).
  • the PCR analysis showed that B2 cells had lost the chloroplast genome.
  • B2 and B3 had the same 6-bp deletions in the Eg- GSL2 gene, and were siblings, their chloroplasts were differently inherited.
  • B2 had no chloroplasts, whereas B3 cells showed underdeveloped chloroplasts compared to the original wild type CK.
  • the difference between B2 and B3 may have been due to asymmetric cell divisions that were observed between B2 and B3 cell lines.
  • Euglena gracilis B2 and B3 cell lines can be grown at large scale in heterotrophic and mixotrophic conditions in combination with aerobic or anaerobic treatments.
  • the B2 cell line can do so without producing any associated chloroplast proteins under light conditions.
  • B2 and B3 can be used for product innovation as well as fundamental research.
  • the development of the B2 cell line that has lost the capacity to make chlorophyll under light condition has important commercial implications. This trait is beneficial to utilize for large scale/open pool fermentation because the B2 cells would no longer need to be cultured in the dark during the fermentation process to prevent production of proteins involved in photosynthesis instead of proteins of interest.
  • the original check strain, or wild type cells showed about 20% more biomass by weight when compared to that of the B2 cell line (using glucose supplemented growth media, pH 3.2).
  • the media was optimized for the wild type (WT) cells.
  • paramylon-free cell line B2 had very high protein (-68% of dry weight) and oil (>18% of dry weight), and very low carbohydrates (3% of dry weight) compared to the wild type WT (Table 2).
  • B2 showed an approximately 18% increase in protein, 59% increase in fat, and an 87% reduction in carbohydrates when compared with the profiles of the wild type CK E. gracilis strain Z cells (Table 2).
  • Table 2 shows the proximate analysis report (SGS, Geneva, Switzerland) of the B2 cell line vs wild type Euglena gracilis strain Z (WT) cultured under heterotrophic/aerobic conditions.
  • the B2 cell line produced significantly higher levels of proteins and lipids but much less carbohydrate than CK.
  • the new paramylon-free cell line (B2) biomass harvested from aerobic condition exhibits high amounts of protein (>71% of dry weight), high oil content ( ⁇ 18% of dry weight), and very low amounts of carbohydrates (1% of dry weight) as shown in Tables 5 and 6 below.
  • the B2 biomass harvested from anaerobic conditions produced much more oil ( ⁇ 30% of dry weight) and more myristic acid (48.1% vs. 35.9%) and produced double the amount of saturated fatty acids (20.1% vs. 10%) compared with the aerobic condition cultured cells (Table 3 and Table 4).
  • Table 3 shows the proximate analysis report from SGS - B2 cells cultured at heterotrophic/aerobic vs heterotrophic/anaerobic conditions. Under aerobic conditions, B2 cells produced slightly more protein while under anaerobic conditions B2 cells produced more lipids. Table 3: Proximate Analysis of B2 Under Different Growth Conditions
  • Table 4 shows the analytical report (SGS, Geneva, Switzerland) of fatty acid profiles (% of total fatty acids) in B2 cells cultured at heterotrophic/aerobic vs. heterotrophic/anaerobic conditions.
  • B2 cells produced more long chain unsaturated fatty acids such as oleic acid, eicosatrienoic acid, and arachidonic acid under aerobic conditions, but produced more saturated fatty acids such as lauric acid and myristic acid under anaerobic conditions.
  • Table 5 shows the results of an analytical report (SGS, German branch) showing amino acid composition in B2 cells cultured under heterotrophic/aerobic vs. heterotrophic/anaerobic conditions.
  • B2 cells produced higher levels of amino acids under aerobic conditions than under anaerobic conditions, except for the amino acids methionine and tryptophan.
  • B2 had higher S -containing amino acid cysteine under heterotrophic/aerobic growth conditions than under heterotrophic/anaerobic conditions.
  • Table 5 Amino Acid Composition in B2 Cells Under Different Growth Conditions
  • Table 6 shows internal analytics of amino acids profile results (g/100 g sample) where H represents heterotrophic, and M represents mixotrophic, A represents aerobic conditions, and AN represents anaerobic conditions of B2, B3, and WT cells. Across all culture conditions, B2 has higher amounts of most amino acids, but not tryptophan.
  • Amino acid profiles of freeze-dried cell biomass samples were determined and analyzed using an Agilent 1260 Infinity 11 LC with a diode-array detector (HPLC- DAD). Table 6: Amino Acid Profiles in B2, B3 and Wild Type Lines Under Different Growth Conditions
  • Table 7 shows internal analytics of fatty acid profiles in fatty acid weight mg per gram of biomass.
  • H represents heterotrophic
  • M represents mixotrophic
  • A represents aerobic conditions
  • AN represents anaerobic conditions of B2, B3, and wild type (WT) cell lines.
  • the most abundant fatty acid in all cell lines is myristic acid.
  • myristic acid levels in B2, B3, and WT accumulate differently under various conditions in that B2 had the most stable levels of myristic acid while WT demonstrated dynamic levels of myristic acid across culture conditions.
  • Fatty acid profiles were determined through total lipid extraction, methylation, and Fatty Acid Methyl Ester (FAMEs) analysis using a GC-F1D Agilent 7890B system.
  • Table 7 Fatty Acid Profiles in B2, B3 and Wild Type (WT) Lines Under Different
  • a wild type (WT) control cell line was used to compare the compositional changes under four different culture conditions: i) Heterotrophic and aerobic fermentation; ii) Heterotrophic and anaerobic fermentation; iii) Mixotrophic and aerobic fermentation; iv) Mixotrophic and anaerobic fermentation.
  • Klebs, ATCC 12894) and the new cell lines B2, B3 were grown in growth media (pH3.2) and were maintained in the lab under dark, heterotrophic culture conditions. Briefly, E. gracilis cells were incubated at 28°C with shaking (120 rpm) and were sub-cultured weekly to maintain as seed cells for inoculations.
  • the same type of glucose supplemented growth media (pH 3.2) is used;
  • the cells are cultured in 1.0 liter of growth media in 2.0 liter of flasks in the INFORS HT Multitron Incubators with Internally installed full spectrum light panel at the light strength of 1600 pmol.
  • the Incubation shakers are set at constant 28°C and 120 rpm.
  • the shakers are covered with aluminum sheet from any light source.
  • the regular stream of nitrogen gas from the compressed N2 cylinder was pumped into the flasks for 2 minutes, then cells were cultured for 48 hours after pumping in with N2 gas.
  • the B2 and WT cells are cultured in 2.0 liter of growth media with 25% more glucose in 3.0 liter of flasks with large vent caps.
  • the purity of paramylon can be determined by the ASC method following the steps: 1) adding paramylon, optionally about 0.5 g, and a magnetic bar to an empty centrifuge tube; 2) adding deionized water, optionally at a ratio of about 50 mL per gram paramylon, into the tube, and stirring for > 8 hours at room temperature; 3) sedimenting the stirred sample by centrifugation, optionally at about 4,700 x g for about 10 min, and decanting supernatant after centrifugation; 4) adding SDS solution, optionally 2% SDS solution, optionally equal volume as the deionized water, to the pellet, and heating the tube at about 110 °C, optionally in an oil bath, for about 30 min with stirring; 5) sedimenting the stirred sample by centrifugation at about 4,700 x g for about 10 min, and decanting supernatant after centrifugation; 6) repeating steps 4-5; 7) adding 70% isopropyl alcohol, optionally at equal volume as the
  • Amino acid profiles were generated using a standard laboratory protocol developed by Agilent Technologies. Total amino acids were extracted through acid and alkaline hydrolysis, external and internal standards were used for quantitative and qualitative analysis. Samples for amino acid profiles were run using an Agilent HPLC- 1260 Infinity II LC with a diode-array detector (HPLC-DAD) and an InfinityLab Poroshell 120 HPH-C18 4.6 x 100 mm, 2.7 mhi column. The DAD detector was set to 262 nm and 338 nm. The injection volume of each sample was 0.5 pL.
  • the autosampler combines Agilent Borate Buffer (0.4 N in water, pH 10.2), Agilent OPA and FMOC reagents and injection diluent (50mL mobile phase A and 0.2 ml. phosphoric acid) with the sample according to the injector program specific to the method.
  • Agilent Borate Buffer 0.4 N in water, pH 10.2
  • Agilent OPA and FMOC reagents
  • injection diluent 50mL mobile phase A and 0.2 ml. phosphoric acid
  • the mobile phase was a solvent gradient of Mobile phase A (10 mM Na2HP04, 10 mM Na2B407, 0.5 mM NaN3 pH 8.2) and Mobile phase B (Acetonitrile / Methanol / Milli Q H20 (45/45/10)) with a flow rate of 1.5 ml./min and heated to a column oven temperature of 40°C.
  • Mobile phase A 10 mM Na2HP04, 10 mM Na2B407, 0.5 mM NaN3 pH 8.2
  • Mobile phase B Alcohol / Methanol / Milli Q H20 (45/45/10)
  • Total lipid extraction was performed using an internal lipid extraction standard method that relies on chloroform and methanol to form a monophasic solvent system to extract and dissolve lipids.
  • a biphasic system is then produced in a purification step by the addition of water leading to the separation of polar and nonpolar compounds.
  • the phases are then separated and the chloroform layer undergoes two washes with 5% sodium chloride.
  • the lipid concentration was determined by evaporating off the chloroform and calculating the remaining mass.
  • FAMEs Fatty Acid Methyl Ester
  • AOAC Official Method 996.06 Following lipid extraction, FAMEs were produced through methylation. Glyceryl triundecanoate heptane solution is added to each sample, after which the heptane is removed using nitrogen (N2) flow to prevent oxidation, and glyceryl triundecanoate is left behind to act as an internal standard. Samples were hydrolyzed with alcoholic NaOH, and methylated with Boron Trifluoride solution (BF3 MeOH). FAMEs were extracted with heptane, and a NaCl solution added to facilitate the separation of layers.
  • the top/heptane layer was acquired and sodium sulphate anhydrous was used to remove water from the samples prior to GC analysis on the Agilent 7890B GC-FID.
  • the sample is injected into the GC where the carrier gas (Helium) transports it through the column and the various components are separated based on their chemical properties.
  • the Flame Ionization Detector (FID) detects the ions formed during the combustion of organic compounds in a hydrogen flame. The generation of these ions is proportional to the concentration of organic species in the sample gas stream.
  • the FAMEs within the sample have a known retention time and therefore we can identify the individual fatty acids and quantify based on peak area. This is the most widely used lipid extraction method and is reliable for lipid concentrations of 2 percent or less.
  • Table 9 Compositional comparison between wild type cell line and B2 cell line measuring: Moisture, Protein, Fat, Ash and carbohydrates (Paramylon) content.
  • Table 10 Total Amino Acid in B2, B3, WT Cell Lines Under Various Culture Conditions. Total Amino acids (g per lOOg of dried biomass) of B2, B3 and WT cell lines under Heterotrophic Aerobic, Heterotrophic Anaerobic, Mixotrophic Aerobic, and Mixotrophic Anaerobic conditions. Standard error is represented by the ⁇ number.
  • Both B2 and B3 produce more all types of amino acids, more essential amino acids, and more total pure proteins than the WT cells (Table 11, FIG. 5).
  • Table 11 Average individual amino acid mass (g) per lOOg of dried biomass for B2, B3, and WT cells grown under heterotrophic and aerobic condition. Standard error is represented by the ⁇ number.
  • both B2 and B3 still produce more all types of amino acids, more essential amino acids, and more total pure proteins than the WT cells (Table 12, FIG. 6).
  • Table 12 Average individual amino acid mass (g) per lOOg of dried biomass for B2, B3, and WT cells grown under heterotrophic and anaerobic condition. Standard error is represented by the ⁇ number.
  • B2 has less protein under light
  • B3 keeps constant under both dark and light
  • the WT has much more amino acids and pure protein accumulated under light.
  • B2 and B3 still produce more all types of amino acids, more essential amino acids, and more total pure protein than the wild type cells under the mixotrophic and aerobic conditions (Table 13, FIG. 7).
  • Table 13 Average individual amino acid mass (g) per lOOg of dried biomass for B2, B3, and WT cells grown under mixotrophic and aerobic condition. Standard error is represented by the ⁇ number.
  • Table 14 Average individual amino acid mass (g) per lOOg of dried biomass for B2, B3, and WT cells grown under mixotrophic and anaerobic condition. Standard error is represented by the ⁇ number.
  • Eg-GSL2 In consistence with our observations of the cells under microscope and the genotyping prediction based on the genomic DNA deletion of a 6 bp fragment in the Eg- GSL2 gene caused by the RNP complex delivered CRISPR gene editing of the paramylon synthase gene, Eg-GSL2, the paramylon content are at zero/free or close to zero in B2 and B3 cells cultured under all the four conditions; While for the wild type Euglena gracilis strain Z, the highest paramylon was accumulated under the heterotrophic and aerobic condition; Light condition reduces the paramylon productivity in the WT cells; Anaerobic condition further dramatically decreases the accumulation of paramylon in the WT cells.
  • the analytical results of the total lipids indicate that the anaerobic condition favors the total lipids accumulation for all the cell lines of the Euglena gracilis.
  • the B2, B3, and WT cell lines exhibit very characteristic patterns with total lipid levels under various conditions
  • the heterotrophic aerobic conditions and mixotrophic anaerobic conditions showed the opposite effects on the total lipids accumulations in the B2, B3, and WT cell lines.
  • the lipids contents are in the order of B2>B3>WT; while under the mixotrophic and anaerobic conditions, the lipids contents are WT>B3>B2 Interestingly B3 sets in between of B2 and WT.
  • HA Heterotrophic/Aerobic
  • HN Heterotrophic/Anaerobic
  • MA Mixotrophic/Aerobic
  • MN Mixotrophic/Anaerobic
  • Table 17 Fatty acid profile of Even and Odd numbered carbon fatty acids in B2, B3 and WT cell lines under Heterotrophic Aerobic condition. Only fatty acids with greater than 0.5 mg/g of freeze-dried biomass is shown. Standard error is represented by the ⁇ number.
  • Table 18 Fatty acid profile of Even and Odd numbered carbon fatty acids in B2, B3 and WT cell lines under Heterotrophic Anaerobic condition. Only fatty acids with greater than 0.5 mg/g of freeze-dried biomass is shown. Standard error is represented by the ⁇ number.
  • Table 19 Fatty acid profile of Even and Odd numbered carbon faty acids in B2, B3 and WT cell lines under Mixotrophic Aerobic condition. Only faty acids with greater than 0.5 mg/g of freeze-dried biomass is shown. Standard error is represented by the ⁇ number.
  • Table 20 Fatty acid profile of Even and Odd numbered carbon fatty acids in B2, B3 and WT cell lines under Mixotrophic Anaerobic condition. Only fatty acids with greater than 0.5 mg/g of freeze-dried biomass is shown. Standard error is represented by the ⁇ number.
  • the anaerobic conditions favor the accumulations of the myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:l), and the medium odd carbon chain fatty acid, tridecylic acid (Cl 3:0) in the most of these experiments; While the light conditions (MA & MN) favor the accumulations of very long chain unsaturated fatty acids such as dihomo-gamma linolenic acid (C20:3) and eicosapentaenoic acid (EPA, C20:5) and the very long chain odd carbon number saturated fatty acid, tricosylic acid (C23:0) (Table 17-20, FIG. 9 and FIG. 10).
  • the most abundant fatty acid from the cells cultured under all the 4 conditions is myristic acid (C14:0); and the myristic acid content peaks in the samples of the cell lines grown under the heterotrophic and anaerobic (HN) condition.
  • the myristic acid is much lower in the new cell lines, B2 and B3, than in the WT cells under anaerobic conditions (HN & MN), but the myristic acid in B2 and B3 cells are higher than in the WT cells under mixotrophic and aerobic condition (MA).
  • the light exposure and anaerobic treatment have the opposite effects compared with the dark and aerobic condition on the fatty acid accumulations.
  • RNA extractions Approximately 0.5 x 10 6 cells/mL to 30 x 10 6 cells/mL were collected over the full cell culture time course for high quality and high purity total RNA extraction using the RNeasy Plus Universal Mini Kit (Catalog no. 73404). For each time point and each cell line, 3 biological replicates were prepared all in the same concentration of 50 ng/ pL. Total of 500 ng RNA for each replicate were used for reverse transcription and cDNA synthesis.
  • cDNA synthesis for gene expression was conducted using the Qiagen QuantiTect Reverse Transcription Kit according to the manufacturer's protocol and qRT-PCR was performed using the QuantStudioTM 3 Real- Time PCR System (Life Technologies). For each qRT-PCR reaction, the reaction volume is in 20 pL, 5 ng of cDNA is used, 1 pL of each gene-specific forward and reverse primers in 10 pM were added, 10 pL of 2 X PowerUp qRT-PCR SYBR Green Master Mix Buffer (Including ROX as the internal passive fluorescent dye) (QIAGEN) were added before starting the qRT-PCR.
  • the qRT-PCR thermo-cycling conditions include the following stages and steps: Holding Stage: 50°C, 2 minutes; 95°C, 2 minutes; Cycling Stage: 40 cycles of 95°C for 3 seconds; 60°C for 30 seconds (Collect SYBR green fluorescence signal during the extension stage at this 60°C temperature); Melt Curve Stage: 95°C, 15 seconds; 60°C, 1 minutes; 95°C, 15 seconds (Collect SYBR green fluorescence signal during the dissociation stage from 60°C to 95°C).
  • the DDO is obtained based on the DO value of a tested sample against the ACt value of a check (CK) at a certain time point, and the actin gene is used as endogenous control gene.
  • the relative gene expression ratio is calculated with the equation of 2 L -DDO.
  • Chloroplast specific gene s expression under mixotrophic and aerobic conditions
  • the cytochrome c synthesis gene ( Chl-ccsA ) is a chloroplast specific chlorophyll synthesis gene. Since B2 lost chloroplast, therefore, no transcript of Chl-ccsA gene is detected in B2 cells over the time course. However, B3 cells reached the highest expression level at Day 6, and WT peaked at Day 4. The results match the cell growth observations that B3 turns green slower than the WT under light (FIG. 12).
  • GAPDH glyceraldehyde-3 -phosphate dehydrogenase
  • GAPDH glyceraldehyde-3 -phosphate dehydrogenase
  • the paramylon degradation gene, Egcell7A encodes an endo-B-1,3- glucanase in Euglena gracilis.
  • Our results showed that the WT cells have relatively higher expression levels of the Egcell 7 A gene than the paramylon free cell lines, B2 and B3, during the early time points (Day 0 and 1) over the time course, while WT has lower expression of the gene during the late growth stages (Day 4 and 6) (FIG. 14).
  • the paramylon degradation gene, Egcell 7 A keeps making transcripts in B2 and B3 cells.
  • Egcell 7 A gene Over the entire time course of the mixotrophic and aerobic condition, the expression of Egcell 7 A gene is relatively stable in B2 and B3 cell lines than in the WT. In a separate gene expression investigation of the paramylon genes, we detected that Egcell 7 A gene has much higher expression levels than all the other paramylon genes.
  • Anaerobic energy metabolism and dark specific protein (Eg-PNO, Pyruvate: NADP+ Oxidoreductase) gene ’s expression under mixotrophic and aerobic conditions
  • pyruvate :NADP+ oxidoreductase functions for the oxidative decarboxylation of pyruvate in the mitochondria.
  • Pyruvate :NADP+ oxidoreductase is involved in the anaerobic energy metabolism.
  • the Eg-PNO protein is specifically accumulated in the Euglena gracilis cells cultured under dark conditions. Interestingly, under the constant light and aerobic condition, Eg-PNO gene’s transcripts were detected through the time course of the mixotrophic and aerobic culture experiment (FIG. 15).
  • Eg-PNO The expression levels of Eg-PNO decline over the time of light exposure from Day 0 to Day 4, and the changes in WT cells are more dramatic than in the B2 and B3 cells. Since the B2 and B3 cells consume less simple carbon source and multiply slowly in comparison with the WT cells, the relative higher expression of Eg-PNO gene is still detected in B2 and B3 cells on Day 6. The results indicate that Euglena gracilis cells make mRNA transcripts of the Eg-PNO gene under constant light condition for the dark specific protein, but once the cells are moved to a dark condition, the mRNA transcripts will be available and get the cells ready to make Eg-PNO protein in the dark condition.
  • Chloroplast specific gene s expression under heterotrophic and aerobic conditions
  • the chloroplast specific chlorophyll synthesis gene, Chl-ccsA is not detected in B2 cells under constant dark conditions, the same as under constant light conditions. Because B2 cells lost chloroplast and there will be no transcript of Chl-ccsA gene produced in B2 cells. However, the Chl-ccsA gene in both B3 and WT cells reached the highest expression level at Day 6 under dark (FIG. 16).
  • the phytoene synthase gene, Eg-crtB, in Euglena gracilis encodes the enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway by the condensation of two molecules of the geranylgeranyl pyrophosphate (GGPP, C20) to the first carotene, phytoene (C40). Suppression of Eg-crtB gene can causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures.
  • Cyclins are eukaryotic proteins that play an active role in controlling nuclear cell division cycles and regulate cyclin dependent kinases (CDKs). Cyclin-dependent kinases interact with cyclins to regulate cell cycle progression and are required for the G 1 and G2 stages of cell division (FIG. 18). Cyclins have no enzymatic activity on their own, but they activate the CDKs by binding to them. Cyclin dependent kinasea are activated when cyclins bind to them. When the cyclin levels decrease, the CDKs become inactive. [0174] Over the time course, the relative expression of the cyclin gene ( Eg-CycA ) in the WT and B2 cells declines after peaked on Day 1.
  • Eg-CycA cyclin dependent kinases
  • the expression pattern in B3 is not as obvious as in B2 and WT cells.
  • the WT has higher expressions of the Eg-CycA gene than B2 and B3; while during the late growing stages (Day 4 and Day 6), B2 has higher expression levels than the WT and B3.
  • the Eg-CycA gene’s expression patterns have high degree of similarities to the phytoene synthase gene, Eg-crtB. Such results indicate that genes involved in cell growth and cell division tend have similar expression patterns or co-expression patterns.
  • CDK-A are relatively stable over the time course (FIG. 19).
  • Genomic DNA fragments including the partial upstream region of Eg-
  • GSL2 gene and partial downstream region of Eg-GSL2 , s start codon were obtained using the gene-specific primer pairs of Eg-GSL2-5U-Fl: 5'-GTCCAATCCCACTCTGAAAGTT-3'
  • Eg-GSL2-5U-R 5'-CATATGGCTTTTCGGATGG-3' (SEQ ID NO:48).
  • the primer pairs were designed based on publicly available whole genome sequencing assembly and whole genome transcriptomes databases.
  • the genomic PCR products of ⁇ 640 bp long were isolated and purified using the QIAquick PCR Purification Kit and the purified PCR product was sequenced using Sanger Sequencing at The Centre for Applied Genomics (TCAG; Toronto).
  • Euglena gracilis Eg-GSL2 Promoter Sequence Identification [0177] From the genome of wild type Euglena gracilis strain Z, a 642 bp fragment of the genomic sequence was obtained and verified. The nucleotide sequence of the 642 bp fragment is SEQ ID NO:49.
  • the sequence includes 375 bp upstream of the start codon and 264 bp downstream of the start codon.
  • the start codon, ATG of Eg-GSL2 is indicated in bold font.
  • the 60 bp fragments before and after the ATG were used as homology arms for precise in-frame CRISPR knock-in of the coding region for genes of interest.
  • This sequence included a 375 bp upstream (partial promoter) of the start codon of Eg-GSL2 (SEQ ID NO:50).
  • a CRISPR guide RNA was designed to the target site around the start codon of the Eg-GSL2 gene, and the 60 bp fragments before and after the start codon (ATG) of Eg- GSL2 gene were used as homology arms for precise in- frame CRISPR knock-in of the genes of interest.
  • the new CRISPR guide RNA target site and knock-in homology arms around the start codon (ATG) of Eg-GSL2 gene were designed in order to introduce coding regions of genes in-frame with Eg-GSL2 gene under its endogenous promoter into the Euglena gracilis genome.
  • the sequence of the CRISPR target sequence is set forth as follows: 5'- GGGGCATTGGAGGGTCATGA - 3' (SEQ ID NO: 13).
  • the CRISPR target sequence designed for precise CRISPR knock-in under the endogenous promoter of the Eg-GSL2 gene had a Doench Activity Score of 0.175, a PAM sequence of AGG, a CRISPR target sequence specificity score of 100.00%, and zero off-target sites against the known publicly available Euglena gracilis genome assembly and whole genome transcriptomes.
  • a total of 6.0 pL of crRNA designed to the target sequence and tracrRNA solution (100 mM) in equal amounts were prepared.
  • the mixture was heated at 95°C for 5 minutes and then cooled at room temperature for 15 minutes.
  • Cas9 Nuclease V3 Integrated DNA Technologies, Coralville, IA
  • ssODN single-stranded donor oligonucleotides
  • the wild type (WT) Euglena gracilis strain Z Euglena gracilis Klebs, ATCC 12894 cells were cultured under heterotrophic and aerobic conditions on an orbital shaker (120 rpm, 28°C) in glucose supplemented growth medium (pH 3.2). After 3 days of growth, cells were collected by centrifugation at 2000 rpm and washed with an electroporation buffer (3:2 of growth medium pH 5.5:0.3M sucrose). Cells were resuspended in the electroporation buffer to obtain a cell concentration of ⁇ 3 x 10 6 cells/ mL.
  • WT wild type
  • Euglena gracilis strain Z Euglena gracilis Klebs, ATCC 12894 cells were cultured under heterotrophic and aerobic conditions on an orbital shaker (120 rpm, 28°C) in glucose supplemented growth medium (pH 3.2). After 3 days of growth, cells were collected by centrifugation at 2000 rpm and washed with
  • a total of 5.0 pL of the RNP- CRISPR/Cas9 + ssODN complex were added to 50.0 pL of the prepared Euglena gracilis cells, and electroporation (NEPA21 Super Electroporator, NEPAGENE, Ichikawa, Japan) was used to introduce the RNP-CRISPR/Cas9 + ssODN complex into Euglena gracilis for targeted in-frame knock-in gene editing. After electroporation, 1.0 mL of growth medium (pH 5.5) was added to the cells, which were incubated for a 48 72 hours in an incubator shaker under heterotrophic and aerobic conditions (120 rpm, 28°C).
  • sequence of the first homology arm (SEQ ID NO:9) is set forth as follows:
  • sequence of the second homology arm (SEQ ID NO: 10) is as follows:
  • ssODN single-stranded oligo DNA nucleotides
  • the nucleic acid molecule of GFP is SEQ ID NO: 11.
  • Receptor binding motif is SEQ ID NO: 12.
  • sequence of the ssODN of GFP (SEQ ID NO: 14) is a single-strand
  • DNA fragment comprising GFP CDS and homology arms.
  • the ssODN of SARS-COV-2 Spike protein/S 1 domain (receptor binding motif underlined) (SEQ ID NO: 15) is a single-strand DNA fragment comprising SARS- COV-2 SP-S1 nucleic acid molecule and homology arms.
  • Euglena gracilis transformants from the RNP treatment of Euglena gracilis with CRISPR in-frame knock-in of single strand DNA of GFP gene have been obtained.
  • Euglena gracilis transformants are shown in bright field and with a light shield observed under the EVOS FL Auto microscope (Life Technologies, Carlsbad, CA). No difference in brightness could be observed among the individual cells and using fluorescent microscopy under a GFP filter and with a light shield under the EVOS FL Auto microscope (Life Technologies, Carlsbad, CA).
  • the brighter cell indicates GFP- expression and positive transformation of Euglena gracilis via RNP-CRISPR mediated in-frame knock-in of single stranded GFP DNA. This is the first known successful precise in-frame knock-in of a nucleic acid molecule resulting in expression of a gene for Euglena gracilis.
  • the expression of heterologous nucleic acid molecules in the Euglena genome can be influenced by the promoter sequences of the endogenous gene.
  • One such promoter sequence useful for expressing heterologous nucleic acid molecules is the Eg- GSL2 promoter (SEQ ID NO: 8).
  • the full-length genomic sequence of Eg-GSL2 including 46 exons and 67,080 base pairs is available from the Brigham Young University Euglena genome assembly contig UTG0016941 (SEQ ID NO:61).
  • the hygromycin resistance and tolerance of the three cell lines is investigated at two different pH levels: 3.2 and 5.5. This is to show how these three cell lines grow or how they are inhibited by the presence of the antibiotic at two different pH levels.
  • the results will help us to understand if the hygromycin resistance is used as a selection marker, what levels of the hygromycin concentration at which pH value are proper for us to set for the selection of any transformants derived from B2 and B3 cell lines and for the selection of any transformants derived from the WT Euglena gracilis strain Z.
  • the Euglena gracilis strain Z ( Euglena gracilis Klebs, ATCC 12894) and the new cell lines B2, B3 were inoculated (60K cells inoculated in each drop, 3 replicates for each cell line) on the solid growth media (15 g agar/L) at pH 3.2 or pH 5.5 supplemented with 50 pg/mL of hygromycin and they were incubated and observed daily until 21 days post-inoculation at room temperature and under regular light condition in the lab.
  • WT cell dots further expanded on the solid growth media with pH 3.2, but not on the solid growth media with pH 5.5. Hygromycin resistance and tolerance tests were performed at pH 3.2 and pH 5.5 for B2, B3 and WT cell lines.
  • the results indicate that the higher the pH value (5.5 vs.
  • the WT E. gracilis cells can grow on the solid growth media at pH5.5 supplemented with hygromycin of 10, 20, 30 pg/mL, while the B2 and B3 cells are much more susceptible to hygromycin, and even the hygromycin of 10 pg/mL can obviously inhibit the growth of B2, and B3 cells in comparison with the WT cells. No colony formed for the B2 and B3 cells on the plate with solid growth media supplemented with the hygromycin of 30 pg/mL.
  • Example 6 First ever successful CRISPR in-frame knock-in of a Gene of Interest (GOI) directly under the endogenous gene expression regulatory elements (both promoter and terminator) of the paramylon degradation gene, Egcell 7A, in Euglena gracilis Introduction:
  • the first successful CRISPR in frame knock in of a gene of interest (GOI) under endogenous regulatory elements (Both Promoter and Terminator) from and in Euglena gracilis is outlined and its endogenous promoter and terminator are utilized for the CRISPR in- frame knock-in of the hygromycin resistance gene (hygromycin phosphotransferase, HPTI1 ), or HygR, short for Hygromycin Resistance (1123 bp including in- frame knock-in homology arms); The Covid-19 Spike protein subunit 1 gene element (SP-S1, 772 bp including in-frame knock-in homology arms); and the green fluorescent protein (mGFP, 826 bp including in-frame knock-in homology arms) gene’s in- frame knock-in under the endogenous gene expression regulatory elements of the paramylon degradation gene ( Egcell 7 A) are also designed, performed, and successfully accomplished with the same approaches as for the hygromycin resistance gene.
  • endogenous regulatory elements Bottom Promoter and Terminator
  • the wild type (WT) Euglena gracilis strain Z ( Euglena gracilis Klebs, ATCC 12894) cells were cultured under heterotrophic and aerobic conditions on an orbital shaker (120 rpm, 28°C) in glucose supplemented growth medium (pH 3.2). After 3 days of growth, cells were collected by centrifugation at 2000 rpm and washed with an electroporation buffer (3:2 of Growth medium pH 5.5:0.3M Sucrose). Cells were resuspended in the electroporation buffer to obtain a cell concentration of ⁇ 2 x 10 6 cells/mL.
  • Single cell selection and cell line development After the recovery period of 48 hours, single cells were isolated using lOx series of dilutions and with the assistance of ZEISS AXIO Vert. A1 microscope. Single cells were maintained in 3 5 pL of modified glucose supplemented growth media (pH 5.5) on a 96-well plate, and were continually monitored. Once a single cell was obtained, 200 pL of modified glucose supplemented growth media was added to the well to allow for growth and cell replication. It takes up to 20 generations to produce ⁇ 1 million cells from a single cell.
  • the sequencing results enable us to read a confirmed sequence of 1137 bp including the 5’ border on the upstream of the start codon of the Egcell7A gene and the 3’ border on the downstream of the stop codon of the Egcell7A gene. Only one single nucleotide differs at the 318 bp (a T to G change) after the start codon between the purified PCR product and the synthesized donor ssODN template.
  • the sequencing results further confirmed that the in- frame knock-in was located in the Egcel 17A- 1 gene between the Egcel 17A- 1 ’s promoter and terminator, the endogenous regulatory elements in Euglena gracilis.
  • the one single nucleotide change does not alter the coding for the amino acid of leucine that locates on the 106 AA of the hygromycin phosphotransferase protein.
  • PCR amplification on the extracted DNA with primers for the CRISPR knock-in sites detection molecular marker was applied to detect the in- frame knock-in of the mGFP gene and the SP-S1 domain in the treated cells.
  • the forward primer of the marker locates on the upstream of both the left arm and the start codon of the Egcell 7 A gene, while the reverse primer of the marker locates on the downstream of both the right arm and stop codon of the Egcell 7 A gene.
  • the PCR was done using the Phire Hot Start Master Mix.
  • the PCR products in the expected sizes for the knock-in of mGFP (864 bp/881 bp) and SP-S1 (810 bp / 827 bp) are amplified from the CRISPR knock-in treated cells, but not from the checks (FIG. 2 IB).
  • Single cell lines carrying the CRISPR in-frame knock-in genes are under selections and development.
  • HPT Hemamycin Phosphotransferase
  • SC2 CRISPR In-frame knock-in cell line
  • HPT-specific monoclonal antibody from abbexa abx018357
  • the immunoblotting was conducted with the primary Mouse Monoclonal HPT Antibody diluted as 1:1000 and the Goat anti-Mouse IgG Secondary Antibody (HRP conjugated) diluted as 1:2000.
  • the expressed protein of the CRISPR in-frame knock-in hygromycin phosphotransferase having molecular weight of 39.25 KDa is detected under dark without hygromycin treatment and under light with higher dose of hygromycin (hygromycin is light sensitive) treatment.
  • the homozygous CRISPR in-frame knock-in SC2 cell line was able to grow in nutrient containing media that does not contain hygromycin, and still express the hygromycin resistance gene. This result indicates that the CRISPR in- frame knock-in gene Hygromycin phosphotransferase is expressed constitutively not induced by adding hygromycin. This result also confirmed that the CRISPR knock-in of the phosphotransferase gene in the genome is permanent in the SC2 cell line under the Egcell 7 A promotor and terminator.
  • the expressed protein of the CRISPR in-frame knock-in hygromycin resistance gene having molecular weight of 39.25 KDa was detected under dark without hygromycin treatment and under light with higher dose of hygromycin conditions.
  • the heterologous protein expression of a Gene of Interest (GOI) under the endogenous promoter and terminator of the paramylon degradation gene ⁇ Egcell 7 A) through the CRISPR in-frame knock-in approach is the first ever successful breakthrough accomplishment in Euglena gracilis.
  • Egcell 7 A Three paramylon degradation genes ⁇ Egcell 7 A, EgceWlA, EgceWlB ) cDNA sequences coding for endo-l,3-beta-glucanase are available from the NCBI GenBank database as listed here.
  • Our gene expression investigation results indicate that Egcell 7 A is highly expressed and Egcell 7 A is the functional paramylon degradation gene, while EgceWlA and EgcelS 1 B are the homologs, they have no detected expressions under heterotrophic and aerobic culture conditions operated at Noblegen Inc. (SEQ ID NOs: 62, 63, 64).
  • Egcell 7 A as the gene target for both CRISPR knock-out (paramylon productivity enhancement) and in- frame knock-in of a Gene of Interest
  • Egcell 7A has a potential to be utilized as a strong endogenous promoter to drive the expression of a Gene of Interest. If the Egcell 7 A gene is knock out, the paramylon productivity can be substantially enhanced.
  • Egcell 7A CRISPR Target Sites and guide RNA (gRNA) sequences (SEQ ID NO:
  • the two CRISPR target sites designed for in-frame knock-in under the endogenous gene expression regulatory elements of the Egcell 7 A gene locate at 7 bp downstream of the start codon (ATG) and 26 bp upstream of the stop codon (TAA).
  • the expected deletions were noted as (nnnnnnnnnnnnnnn- 13,008 bp or 15,803 bp, or 13,719 bp) for the Egcell 7 A gene and its homologs.
  • GFP green fluorescent protein gene
  • Hygromycin selection of CRISPR in-frame knock-in complex of the HygR/Hptll gene was performed with 25 pg/mL, 30 pg/mL and 35 pg/mL of Hygromycin.
  • WT cells treated with CRISPR in-frame Knock-in complex of the HygR/Hptll gene have much more resistance colonies formed on the plates with 25, 30, and 35 pg/mL of Hygromycin.
  • Sample 4 B3 cells.
  • Sample 5 Bl l cells.
  • Sample 6 C8 cells.
  • Sample 7 CIO cells.
  • Sample 8 D3 cells.
  • Sample 9 Dl l cells.
  • Sample 10 D12 cells.
  • Sample 11 E5 cells.
  • Sample 12 E12 cells.
  • Sample 13 F3 cells.
  • Sample 14 F6 cells.
  • M represents the ladder
  • ssODN-HygR represents the single-stranded oligodeoxynucleotides of the hygromycin resistance gene donor template used as a positive control for the PCR.
  • PCR gel of a selection of homozygous CRISPR in- frame knock-in cell lines was performed. Out of the six CRISPR in-frame knock-in single colony cell lines, only one homozygous CRISPR in-frame knock-in cell line, SC2, carrying the hygromycin phosphotransferase gene (Hptll/HygR) was obtained.
  • the ssODN-HygR represents the single-stranded oligodeoxynucleotides of the hygromycin resistance gene donor template used as a negative control for the detection of the CRISPR in-frame knock-in sites that are beyond the ssODN-HygR donor template; NTC no DNA template control, WT wild type.
  • SC2 single colony cell line
  • the sequencing results enable us to read a confirmed sequence of 1137 bp including the 5’ border on the upstream of the start codon of the Egcell 7A gene and the 3 ’ border on the downstream of the stop codon of the Egcel 17A gene. Only one single nucleotide differs at the 318 bp (a T to G change) after the start codon between the purified PCR product and the synthesized donor ssODN template.
  • the sequencing results further confirmed that the in- frame knock-in was located in the Egcel 17A- 1 gene between the Egcell7A-l ’s promoter and terminator, the endogenous regulatory elements in Euglena gracilis.
  • the one single nucleotide change (G318T) does not alter the coding for the amino acid of leucine that locates on the 106 AA of the hygromycin phosphotransferase protein. (SEQ ID NOs:87, 88, and 89).
  • Egcell 7 A from Euglena gracilis. Many more and high value-added products can be produced in Euglena gracilis through the CRISPR in-frame knock-in system established by the inventors of this invention using the endogenous gene expression regulatory elements (promoter and terminator) of the paramylon degradation gene, Egcell 7 A, from Euglena gracilis.
  • GAPDH Glyceraldehyde-3 -phosphate dehydrogenase
  • the enzyme glyceraldehyde-3 - phosphate dehydrogenase (GAPDH) is an essential component of the glycolytic pathway and converts glyceraldehyde-3 -phosphate to 1,3-bisphosphoglycerate.
  • GAPDH is also a multi-functional and abundant protein. Further investigations to exploit the usefulness of the GAPDH promoter and its interaction/regulatory networks will be fundamentally important to gain a better understanding of the metabolite composition in B2, B3, and WT cell lines.
  • Example 7 Genetic Transformation and Introduction of Nucleic Acid Molecules of Genes of Interest
  • a map of the pCAMBIA 1302-GFP vector having green fluorescent protein (“GFP”) contains a CaMV 35S promoter, an mgfp5, a 6xHIS, an NOS terminator, an RB T-DNA repeat, a pVSl StaA, a pVSl RepA, a KanR, a LB T-DNA repeat, a CaMV poly(A) signal, a HygR an enhanced CaMV 35S promoter and an MCS.
  • SARS- CoV-2 template DNA was purchased from CODEX DNA (San Diego, CA).
  • FL-SP Full length spike protein
  • SP-S1 spike protein subunit 1
  • PCR products obtained from using these primers were purified from an agarose gel and ligated into corresponding restriction sites (using Ncol/Pmll for both FL-SP: HR V3 C: 6HIS and SP-S1 :HRV3C:6HIS while using Spel/Ncol for SPI:GFP:6HIS) of the pCAMBIA1302-GFP vector using the In-Fusion kit (Takara Bio. Inc., Shiga, Japan) to generate pCAMBIA1302-SP_Sl-GFP, pCAMBIA1302- FL SP: HRV3 C : 6HIS , and pCAMBIA 1302-SP_S 1 :HRV3 C : 6HIS .
  • the pCAMBIA 1302- SP_S1 vector contains a CaMV 35S promoter, a SP-S1, a mgfp5, a 6xHIS, a NOS terminator, a RB T-DNA repeat, a pVSl StaA, a pVSl RepA, a KanR, a LB T-DNA repeat, a CaMV poly(A) signal, a HygR, an enhanced CaMV 35S promoter, and a MCS.
  • the pCAMBIA1302-FL_SP:HRV3C:6HIS vector contains a CaMV 35S promoter, a SARS-CoV-2 Spike Protein, a HRV 3C Site, a 6xHIS, a NOS terminator, a RB T-DNA repeat, a pVSl StaA, a pVSl RepA, a KanR, a LB T-DNA repeat, a CaMV poly(A) signal, a HygR, an enhanced CaMV 35S promoter, and a MCS.
  • the pCAMBIA1302- SP_S1:HRV3C:6HIS vector contains a CaMV 35S promoter, a HRV 3C Site, a 6xHIS, a NOS terminator, a RB T-DNA, a pVSl StaA, a pVSl RepA, a KanR, a LB T DNA, a CaMV poly(A) signal, a HygR, an enhanced CaMV 35S promoter, and a MCS.
  • Euglena gracilis Cell Line B2 had a Unique Sensitivity to Antibiotics [0244] Initially, the wild type Euglena gracilis strain Z was found to be able to grow and survive temporarily on high doses of hygromycin (50 pg/mL) at pH3.2 on agar plates rendering them unable to be used for selecting transformants. However, it was found that growth of B2 cells was more obviously inhibited on hygromycin at 50 pg /ml. However, none of the cells of B2, B3, and WT could survive from the high dose of hygromycin (50 pg/mL) at pH 5.5 after 1 week as observed on the 8th Day Post- Inoculation.
  • hygromycin 50 pg/mL
  • hygromycin A series of concentrations of hygromycin at pH5.5 were used to investigate its growth inhibition effects on B2 and the use of the B2 cell line to select for transformants.
  • Transgenic cassettes including the hygromycin resistance gene and the spike protein SI subunit (SP-S1) fused with GFP were successfully transformed into the B2 cell line using electroporation.
  • a linear 5,120 bp PCR product that extended from the left border to the right border of the T-DNA was used.
  • This PCR product contained the transgenic cassettes of (1) the enhanced CaMV 35 S promoter and hygromycin resistance gene and (2) the CaMV 35S promoter and SP-S1 fused with GFP.
  • the PCR product was amplified from the plasmid of pCAMBIA1302-SP_Sl-GFP using the primer pairs: pCAMBIA-LB-F: ACT GAT GGGCT GCCT GTAT C (SEQ ID NO:59) and pCAMBIA- RB-R: CACATACAAATGGACGAACGGA (SEQ ID NO:60).
  • Vector pCAMBIA1302-SP_Sl-GFP containing transgenic cassettes including the hygromycin resistance gene (HygR) and SARS-COV-2 spike protein SI subunit (SP-S1) fused with GFP was also transformed into the high protein cell line B2 cells using electroporation.
  • B2 transformants with vector pCAMBIA1302-SP_Sl-GFP carrying the cassettes of hygromycin resistance gene and SP-S1+GFP formed positive colonies on 30 pg/mL hygromycin using glucose supplemented growth media, pH 5.5 on agar plates after 2-3 weeks. Untransformed B2 cells did not form any surviving colonies on 30 pg/mL hygromycin after 2-3 weeks of selections.
  • Euglena gracilis B2 cell line transformants with hygromycin resistance and the SARS-COV-2 spike protein SI subunit (SP-S1) fused with GFP were actively growing on plates with 30 pg/mL hygromycin. Continuous and multiple rounds of selections for stable transformants of B2 cells with SP-S1 protein are ongoing. Genetic transformants with the nucleic acid molecule of the genes of interest for heterologous protein expressions are confirmed with molecular verification approaches. High efficiency heterologous protein expression and corresponding protein purification using the high protein, paramylon-free cell line B2 will be used for high value-added products such as vaccine production.
  • the high protein and paramylon-free cell lines have been successfully used for heterologous protein expression of (i) the SARS-CoV-2 Spike Protein Sub-unit- 1 : green fluorescent protein (GFP) fusion, (ii) the SARS-CoV-2 Spike protein subunit- 1 with cleavable HIS tag, and (iii) the full length SARS-CoV-2 Spike protein with cleavable HIS tag.
  • GFP green fluorescent protein
  • the B2 cell line was used for genetic stacking by introducing the gene for green fluorescent protein (GFP) and/or variations of the SARS-CoV-2 spike protein gene into the B2 genome using plasmid based genetic transformation approaches. In all cases, use of the B2 cell line led to recombinant protein production.
  • the WT Euglena gracilis strain Z was also used to introduce the gene encoding GFP and/or variations of the SARS-CoV-2 spike protein gene into using RNP delivered CRISPR/Cas9 and repair templates with homology arms. In all cases, use of RNP-CRISPR/Cas9 and WT Euglena gracilis strain Z led to recombinant protein production.
  • An alternative strategy at expressing a gene of interest into Euglena gracilis is through the transformation of DNA plasmid vectors.
  • the DNA plasmid vector PCAMBIA1302 with either GFP (Green fluorescent protein) or with Covid 19 Spike subunit 1 (SP-S1) was transformed into Euglena gracilis cell lines.
  • PCR amplification of the gene and PCR product sequencing are used to positive transformation and western blotting with a specific primary anybody for SP-S1 is used to confirm protein expression in transformed cell lines.
  • the hygromycin concentration at between 10 30 pg/mL at pH 5.5 were set for the selection of any transformants derived from B2 and B3 cell lines; while the hygromycin concentration at 30 35 pg/mL at pH5.5 were set for the selection of transgenic transformant if the WT cells are used as the host cells.
  • Genomic DNA extraction and PCR product purification and sequencing [0251] For each test, approximately 1 x 10 6 Euglena cells ( ⁇ 10 mg) were collected for high quality and high purity genomic DNA extraction using the Qiagen DNeasy Blood & Tissue Kit (REF# 69504). The gene-specific genomic PCR product corresponding to a target gene’s genomic fragment was isolated and purified using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up Kit (REF No. 740609.50) and the purified PCR product was sequenced using Sanger Sequencing at The Centre for Applied Genomics (TCAG; Toronto).
  • the Novex Bolt Mini Gel Tank and the PowerEase 500 power supply were used for the SDS-PAGE protein gel electrophoresis.
  • a total of 750 pg cell lysate for each of the tested cell lines/clones were loaded onto the protein separation gel (Novex Wedge Well 10-20% Tris-Glycine Gel).
  • the separated proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane using the iBlot 2 Gel Transfer Device.
  • PVDF polyvinylidene difluoride
  • Competent Cells Enough plasmid DNA (10,550 bp) (100 pL x —100 ng/ pL) of the DNA plasmid pCAMBIA1302-GFP was obtained from 6 individual positive clones of the transformed E. coli strain whilr competent cells as exhibited on agarose gel.
  • Transformation into B2, B3, and WT cells Two micrograms (4 pL of 500 ng/ pL) of condensed pCAMBIA1302-GFP, 20 DPI plasmid DNA was used to transform the Euglena gracilis B2, B3, and WT cell lines. Transformed WT, B2, and B3 cells (20 DPI) were grown on 10 pg/mL, 20 pg/mL and 30 pg/mL hygromycin with pCAMBAI1302-GFP. The transformed cells through electroporation were plated on the solid growth media and were observed daily.
  • the transformed B2 and B3 cells can only form colonies on the plates with hygromycin of 10 pg/mL, but not on the plates with higher concentration of hygromycin, while the transformed WT cells can form colonies on all the plates with hygromycin of 10, 20, and 30 pg/mL respectively.
  • Wells 4-8 had positive WT B3 transformants that were selected from the 30 pg/mL plate.
  • the WT/CK well was a negative control that had not be transformed.
  • NTC was another negative control.
  • the HygR/Hptll gene-specific PCR results confirmed that all the survived colonies are positively containing the transgenic element of the HygR/Hptll gene for the PCR results.
  • These positive clones derived from transformed B3 and WT cells are under further observation and characterizations. PCR results showed the positive clones/colonies derived from transformed B3 and WT cells with pCAMBIA1302-GFP.
  • PCR confirmation of Transformants carrying transgenic elements from plasmid pCAMBIAl 302-SP S1 was performed. PCR products corresponding to the 198 bp of the SP-S1 subunit specific marker were amplified from the transformants, H30-3 and H30-6. The sample of H10-13 from the second round of selection is used as a negative control for the SP-S1 marker; the B2 cells and the No-template control (NTC) are used as negative controls as well. PCR products corresponding to the 359 bp of the HygR/Hptll gene-specific marker were amplified from the transformants, H30-3 and H30-6. The sample of HI 0-13 from the second round of selection was used as a positive control for the HygR/Hptll marker; the B2 cells and the No-template control (NTC) were used as negative controls.
  • H30-3 and H30-6 are proved to contain both hygromycin resistance gene (HygR/Hptll) and the SP-S1 protein subunit DNA sequence.
  • Both transformants, H30-3 and H30-6 have the correct PCR products corresponding to the 359 bp fragment for a HygR/Hptll gene-specific marker and the correct PCR product corresponding to the 198 bp SP-S1 protein subunit DNA fragment for this section (PCR was done using the Phire Hot Start Master Mix).
  • the transformants, H30-3 and H30-6 were selected for further verifications.
  • HygR/Hptll gene specific marker (359 bp) and the SP-S1 subunit-specific marker (198 bp) confirmed that the H30-3 and H30-6 are the real transformants carrying both the hygromycin gene (HygR/Hptll) and the SP-S1 protein subunit.
  • the 1st Antibody SARS-CoV-2 Coronavirus Spike Protein Subunit 1 Polyclonal Antibody
  • the 2nd Antibody Goat anti-Rabbit IgG Secondary Antibody, HRP conjugated
  • Example 9 Primers used in gene expression and PCR product confirmation
  • the following primers in Table 21 were used in the Examples above for either gene expression or for cell line and transformant identification (as indicated in the table).
  • Table 21 Gene names and primers used in the examples above

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Abstract

La présente invention concerne les éléments suivants : un organisme microalgal avec une altération génétique non transgénique, induite par l'homme ; un procédé de modification d'un caractère dans un organisme microalgal par une altération génétique non transgénique, induite par l'homme ; un procédé d'expression d'une molécule d'acide nucléique hétérologue dans un génome cible d'un organisme microalgal ; une composition pour exprimer une molécule d'acide nucléique hétérologue dans un site d'insertion du génome cible d'un organisme microalgal ; un procédé d'expression d'une séquence nucléotidique dans un organisme microalgal ; et un procédé de production de protéines, lipides, acides aminés, acides gras dans un organisme microalgal.
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WO2024262951A1 (fr) * 2023-06-20 2024-12-26 씨제이제일제당 (주) Vecteur recombiné pour une expression génique élevée

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