Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems

Eleven published articles (4 reviews, 7 research papers) are collected in the Special Issue entitled “Organelle Genetics in Plants.” This selection of papers covers a wide range of topics related to chloroplasts and plant mitochondria research: (i) organellar gene expression (OGE) and, more specifically, chloroplast RNA editing in soybean, mitochondria RNA editing, and intron splicing in soybean during nodulation, as well as the study of the roles of transcriptional and posttranscriptional regulation of OGE in plant adaptation to environmental stress; (ii) analysis of the nuclear integrants of mitochondrial DNA (NUMTs) or plastid DNA (NUPTs); (iii) sequencing and characterization of mitochondrial and chloroplast genomes; (iv) recent advances in plastid genome engineering. Here we summarize the main findings of these works, which represent the latest research on the genetics, genomics, and biotechnology of chloroplasts and mitochondria.

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High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica

Metabolic Engineering ◽

10.1016/j.ymben.2021.04.012 ◽

2021 ◽

Author(s):

Christian Südfeld ◽

Michal Hubáček ◽

Daniel Rodrigues Figueiredo ◽

Mihris I.S. Naduthodi ◽

John van der Oost ◽

...

Keyword(s):

High Throughput ◽

Lipid Accumulation ◽

Insertional Mutagenesis ◽

Nannochloropsis Oceanica ◽

Novel Targets

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Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

Science Advances ◽

10.1126/sciadv.abd6030 ◽

2021 ◽

Vol 7 (11) ◽

pp. eabd6030

Author(s):

Isabel Strohkendl ◽

Fatema A. Saifuddin ◽

Bryan A. Gibson ◽

Michael K. Rosen ◽

Rick Russell ◽

...

Keyword(s):

Histone Modifications ◽

Dna Cleavage ◽

Genome Engineering ◽

Eukaryotic Cells ◽

Loop Formation ◽

Chromatin Compaction ◽

Dna Targeting ◽

Genomic Regions ◽

Dna Bendability ◽

Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

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Microalgae as Sustainable Bio-Factories of Healthy Lipids: Evaluating Fatty Acid Content and Antioxidant Activity

Marine Drugs ◽

10.3390/md19070357 ◽

2021 ◽

Vol 19 (7) ◽

pp. 357

Author(s):

Tiago A. Conde ◽

Bruna F. Neves ◽

Daniela Couto ◽

Tânia Melo ◽

Bruno Neves ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Activities ◽

Fatty Acid Content ◽

Scenedesmus Obliquus ◽

Food Sources ◽

Gas Chromatography Mass Spectrometry ◽

Nannochloropsis Oceanica ◽

Omega 3 ◽

Bioactive Lipids ◽

Food Ingredients

The demand for sustainable and environmentally friendly food sources and food ingredients is increasing, and microalgae are promoted as a sustainable source of essential and bioactive lipids, with high levels of omega-3 fatty acids (ω-3 FA), comparable to those of fish. However, most FA screening studies on algae are scattered or use different methodologies, preventing a true comparison of its content between microalgae. In this work, we used gas-chromatography mass-spectrometry (GC-MS) to characterize the FA profile of seven different commercial microalgae with biotechnological applications (Chlorella vulgaris, Chlorococcum amblystomatis, Scenedesmus obliquus, Tetraselmis chui, Phaeodactylum tricornutum, Spirulina sp., and Nannochloropsis oceanica). Screening for antioxidant activity was also performed to understand the relationship between FA profile and bioactivity. Microalgae exhibited specific FA profiles with a different composition, namely in the ω-3 FA profile, but with species of the same phylum showing similar tendencies. The different lipid extracts showed similar antioxidant activities, but with a low activity of the extracts of Nannochloropsis oceanica. Overall, this study provides a direct comparison of FA profiles between microalgae species, supporting the role of these species as alternative, sustainable, and healthy sources of essential lipids.

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