CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis.
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ABSTRACT:
SUBMITTER: Ishikawa M
PROVIDER: S-EPMC9616515 | biostudies-literature | 2022 Nov
REPOSITORIES: biostudies-literature
Publications
CRISPR/Cas9-mediated generation of non-motile mutants to improve the harvesting efficiency of mass-cultivated Euglena gracilis.
Ishikawa Marumi M Nomura Toshihisa T Tamaki Shun S Ozasa Kazunari K Suzuki Tomoko T Toyooka Kiminori K Hirota Kikue K Yamada Koji K Suzuki Kengo K Mochida Keiichi K
Plant biotechnology journal 20220908 11
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Project description:Euglena gracilis is a flagellate photosynthetic microalga that, thanks to its metabolic adaptability, can grow under both autotrophic and heterotrophic conditions. We focused on the proteome of E. gracilis cultivated in Cramer-Myers medium in dark and light conditions. Cultures grown in the light showed a characteristic green coloration, while cultures incubated in the dark were bright, almost colorless, and had 60% fewer cells per volume. When cultured in the dark, microalga showed reduced concentration of chlorophylls (a, b, and total) and carotenoids compared to cells cultured in the light. Conversely, there was an increase in proline content in the dark compared to light cultivation. Phenol-extracted proteins were digested and purified using the SP3 protocol. We revealed 162 proteins, classified into 12 functional groups, which differentially accumulated in light- and dark-grown cells. The most represented categories in the case of dark cultivation were defense mechanisms, metabolism, and energy. Of note, in the category metabolism of lipids and sterols, we detected 4 proteins involved in the β-oxidation of fatty acids more abundant in the dark. Accumulation os stress- and detoxification-related proteins in microalgal cells cultivated in darkness supported interpretation of such condition as challenging stress factor. Euglena gracilis cultured in the dark is useful for basic research, applied biotechnology, and environmental protection.
2025-03-06 | PXD058143 | Pride
Project description:BackgroundSingle-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency.ResultsHere, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes.ConclusionsBy a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.
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Project description:Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.
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Project description:The current commonly used single-guide RNA (sgRNA) structure has a shortened duplex compared with the native bacterial clustered regularly interspaced short palindromic repeats RNA (crRNA)–transactivating crRNA (tracrRNA) duplex. Here we show that modifying the sgRNA structure by extending the duplex length and mutating the fourth T of the continuous sequence of Ts (which is the pause signal for RNA polymerase III [pol III]) to C or G significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the new sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss-of-function in non-coding genes.
2015-11-17 | GSE74766 | GEO
Project description:Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.
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Project description:Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.
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Project description:Dietary supplementation with nutrients able to control intestinal and systemic inflammation is of marketable interest. Indeed, gastrointestinal homeostasis plays a significant role in maintaining human health. In this setting, E. gracilis may sustain or promote human health, but the effects on the intestinal inflammatory milieu are not clear. In this study, we investigated the anti-inflammatory activity of E. gracilis and inferred possible mechanisms. Paramylon, crude, and fractionated extracts were obtained from E. gracilis grown in vitro. Phytoconstituents of the extracts were characterized using TLC and HPLC UV-Vis. The anti-inflammatory and antioxidant activities were investigated in primary human macrophages and an intestinal epithelial cell line (HT-29). The analysis of the extracts led to identifying β-carotene, neoxanthin, diadinoxanthin, canthaxanthin, and breakdown products such as pheophytins and pheophorbides. E. gracilis fractionated extracts reduced the production of tumor necrosis factor-α triggered by bacterial lipopolysaccharide (LPS) in the short and long terms. Pheophytin a and b and canthaxanthin increased the intracellular reducing potential and dampened the production of LPS-induced reactive oxygen species and lipid peroxidation, intracellular events usually involved in the perpetuation of chronic inflammatory disorders. This study rationalizes the role of specific extract fractions of E. gracilis in controlling LPS-driven intestinal inflammation.
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Project description:Euglena gracilis Transcriptome or Gene expression
| PRJNA1049904 | ENA