Biology subjects

Xin, Y.

Publications and source records attributed to Xin, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the /{beta} hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells. Significant statementLipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative /{beta}- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.

biochemistry↗

bioRxiv2023

Patient-Specific Analysis of Co-expression Networks for Predicting Clinical Outcomes in Breast Cancer

To effectively understand the underlying mechanisms of disease and inform the development of personalized therapies, it is critical to harness the power of differential co-expression network analysis. Despite the promise of differential co-expression network analysis in precision medicine, current approaches have a major limitation: they measure an average differential network across multiple samples, which means the specific etiology of individual patients is often overlooked. To address this, we present Cosinet, a differential co-expression-based single-sample network rewiring degree quantification tool. By analyzing two breast cancer datasets, we demonstrate that Cosinet can identify important differences in gene co-expression patterns between individual patients and generate scores for each individual that are significantly associated with overall survival, recurrence-free interval, and other clinical outcomes, even after adjusting for risk factors such as age, tumor size, HER2 status and PAM50 subtypes. Cosinet represents a remarkable development toward unlocking the potential of differential co-expression analysis in the context of precision medicine.

bioinformatics↗

bioRxiv2023

Structural insights into DNA Topoisomerase II of African Swine Fever Virus

Type II topoisomerases are ubiquitous enzymes that play a pivotal role in modulating the topological configuration of dsDNA. These Topo enzymes are indispensable for DNA metabolism and have been extensively investigated in both prokaryotic and eukaryotic organisms. However, understanding of virus-encoded Topo II remains limited. One intriguing example is the African swine fever virus (ASFV), which stands as the sole mammalian-infecting virus encoding a Topo II. In this work, we utilized multiple approaches involving cryo-EM, X-ray crystallography, atomic force microscopy and biochemical assays to investigate the structure and function of ASFV Topo , pP1192R. We determined the structures of pP1192R in different conformational states and confirming its enzymatic activity in vitro. Collectively, our results unveil the basic mechanisms of viral Topo II, thereby not only augmenting our understanding of this kind of intriguing enzymes but also presenting a potential avenue for the intervention strategies to mitigate the impact of African swine fever virus.

molecular biology↗

bioRxiv2023

Using graph-based model to identify cell specific synthetic lethal effects

Synthetic lethal (SL) pairs are pairs of genes whose simultaneous loss-of-function results in cell death, while a damaging mutation of either gene alone does not affect the cells survival. This makes SL pairs attractive targets for precision cancer therapies, as targeting the unimpaired gene of the SL pair can selectively kill cancer cells that already harbor the impaired gene. Limited by the difficulty of finding true SL pairs, especially on specific cell types, the identification of SL targets still relies on expensive, time-consuming experimental approaches. In this work, we utilized various cell-line specific omics data to design a deep learning model for predicting SL pairs on particular cell-lines. By incorporating multiple types of cell-specific omics data with a self-attention module, we represent gene relationships as graphs. Our approach demonstrates the potential to facilitate the discovery of cell-specific SL targets for cancer therapeutics, providing a tool to unearth mechanisms underlying the origin of SL in cancer biology. Our approach allows for prediction of SL pairs in a cell-specific manner and enhances cancer precision medicine. The code and data of our approach can be found at https://github.com/promethiume/SLwise HighlightsO_LIFew computational methods can systematically predict SL pairs at a cell-specific level, and their performance may not generalize well to clinical scenarios due to the heterogeneity of cancer types. C_LIO_LIThe SLWise utilizes various cell-line specific omics data to design a deep learning model with a graph-based representation and self-attention mechanism. C_LIO_LIThis approach allows for the prediction of SL pairs in a cell-specific manner, providing valuable insights on effectively identifying the cell-type specific SL targets for personalized treatment strategies. C_LI

bioinformatics↗

bioRxiv2023

Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells

The progressive death of mature neurons often results in neurodegenerative diseases. While the previous studies have mostly focused on identifying intrinsic mechanisms controlling neuronal survival, the extracellular environment also plays a critical role in regulating cell viability. Here we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). Although the direct effect of the ONC is restricted to the RGCs, we observed transcriptomic responses in other retinal cells to the injury based on the single-cell RNA-seq, with astrocytes and Muller glia having the most interactions with RGCs. By comparing the RGC subclasses with distinct resilience to ONC-induced cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with other retinal cells, suggesting that these RGCs are intrinsically programmed to foster more communication with their surroundings. Furthermore, we identified the top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, -opioid receptor (Oprm1), a receptor known to play roles in regulating pain, reward, and addictive behavior. Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGC), its neuroprotective effect could be transferred to multiple RGC subclasses by selectively overexpressing Oprm1 in pan-RGCs in ONC, excitotoxicity, and glaucoma models. Lastly, manipulating Oprm1 activity improved visual functions or altered pupillary light response in mice. Our study provides an atlas of cell-cell interactions in intact and post-ONC retina, and a strategy to predict molecular mechanisms controlling neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival.

neuroscience↗

bioRxiv2023

Carotenoid assembly regulates quinone diffusion and the Roseiflexus castenholzii reaction center-light harvesting complex architecture

Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LH{beta} subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.

biochemistry↗

bioRxiv2022

Somatostatin interneurons in auditory cortex regulate cortical representations and contribute to auditory perception

In the mammalian neocortex, somatostatin (SOM)-expressing GABAergic interneurons serve as a critical circuit component, receiving diverse inputs and targeting various types of cortical neurons. However, their role in perception and cognition remains not well understood. In this study, we employed cell-type specific imaging and perturbation during an auditory perceptual decision task to investigate the contribution of SOM interneurons. Two-photon calcium imaging revealed that auditory cortex (ACx) SOM interneurons exhibit not only frequency-selective responses but also selectivity to categorical choices. Simultaneous optogenetic inactivation and two-photon imaging revealed that SOM interneurons refine the frequency discriminability of auditory cortical neurons and contribute to their selectivity to the categorical decision boundary. Moreover, bilateral chemogenetic inactivation of ACx SOM interneurons impaired behavior-level perceptual categorization. Our findings provide a mechanistic understanding on the functional significance of SOM interneurons in auditory cortical processing and perceptual categorization, linking cell-type specific cortical circuit computations with perceptual functions.

neuroscience↗

bioRxiv2022

Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices

Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.

biophysics↗

bioRxiv2022

Nrf2 prevents diabetic cardiomyopathy via antioxidation and normalization of glucose and lipid metabolism in the heart

ObjectiveMetabolic disorders and oxidative stress are the main causes of diabetic cardiomyopathy. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a powerful antioxidant effect and prevents the progression of diabetic cardiomyopathy. However, the mechanism of its cardiac protection and direct action on cardiomyocytes are not well understood. MethodsIn this study, cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) were used to directly observe whether cardiomyocyte-specific overexpression of Nrf2 can prevent diabetic cardiomyopathy and correct glucose and lipid metabolism disorders in the heart. ResultsCompared to wild-type (WT) mice, Nrf2-TG mice showed resistance to diabetic cardiomyopathy in a streptozotocin (STZ)-induced type 1 diabetes mouse model. This was primarily manifested as improved echocardiography results as well as reduced myocardial fibrosis, cardiac inflammation, and oxidative stress. ConclusionThese results showed that Nrf2 can directly act on cardiomyocytes to play a cardioprotective role. Mechanistically, the cardioprotective effects of Nrf2 depend on its antioxidation activity, partially through improving glucose and lipid metabolism by targeting the metabolic pathways of Akt/GSK-3 {beta}/HK-II and AMPK/Sirt1/PGC-1.

molecular biology↗

bioRxiv2022

LRLoop: Feedback loops as a design principle of cell-cell communication

Genome-wide gene expression profiling, and single-cell RNA-seq in particular, allows the predictions of molecular mechanisms regulating cell-cell communication based on the expression of known ligand-receptor pairs. Currently available techniques for predicting ligand-receptor interactions are one-directional from sender to receiver cells. Here we describe LRLoop, a new method for analyzing cell-cell communication that is based on bi-directional ligand-receptor interactions, where two pairs of ligand-receptor interactions are identified that are responsive to each other, and thereby form a closed feedback loop. We assessed LRLoop using both bulk and single-cell datasets and found our method significantly reduces the false positive rate seen with existing methods. Finally, we applied LRLoop to the single-cell datasets obtained from retinal development and discovered many new bi-directional ligand-receptor interactions among individual cell types that potentially control proliferation, neurogenesis and/or cell fate specification.

bioinformatics↗

bioRxiv2022

A Systematic, Complexity-Reduction Approach to Dissect Microbiome: the Kombucha Tea Microbiome as an Example

One defining goal of microbiome research is to uncover mechanistic causation that dictates the emergence of structural and functional traits of microbiomes. However, the extraordinary degree of ecosystem complexity has hampered the realization of the goal. Here we developed a systematic, complexity-reducing strategy to mechanistically elucidate the compositional and metabolic characteristics of microbiome by using the kombucha tea microbiome as an example. The strategy centered around a two-species core that was abstracted from but recapitulated the native counterpart. The core was convergent in its composition, coordinated on temporal metabolic patterns, and capable for pellicle formation. Controlled fermentations uncovered the drivers of these characteristics, which were also demonstrated translatable to provide insights into the properties of communities with increased complexity and altered conditions. This work unravels the pattern and process underlying the kombucha tea microbiome, providing a potential conceptual framework for mechanistic investigation of microbiome behaviors.

systems biology↗

bioRxiv2021

ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication

RationaleMitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types1-3. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection1, 4. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure1. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models1, 4, 5. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue6, 7. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling. ObjectiveThis study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication. Methods and ResultsUsing high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound {beta}-ACTIN only exists in MAM and not in the purified mitochondria. Additionally, co-immunoprecipitation pulls down both DRP1 and {beta}-ACTIN together. Inhibition of {beta}-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and {beta}-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts. ConclusionsThe results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with {beta}-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through {beta}-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

physiology↗

bioRxiv2021

MK2 Expression Promotes Non-Small Cell Lung Cancer Cell Death and Predicts Survival

Non-small cell lung cancers demonstrate intrinsic resistance to cell death even in response to chemotherapy. Previous work suggested that defective nuclear translocation of active caspase 3 may play a role in resistance to cell death. Separately, our group has identified that mitogen activated protein kinase activated protein kinase 2 (MK2) is required for nuclear translocation of active caspase 3 in the execution of apoptosis. This study demonstrates a relatively low expression of MK2 in non-small cell lung carcinoma cell lines compared to small cell carcinoma cell lines. Further, overexpression of MK2 in non-small cell lung carcinoma cell lines results in increased caspase 3 activity and caspase 3 mediated cell death. Higher MK2 transcript levels were observed in patients with earlier-stage non-small cell lung cancer. Higher expression of MK2 is associated with better survival in patients with early stage non-small cell lung cancer across two independent clinical datasets. Using data sets spanning multiple cancer types, we observed improved survival with higher MK2 expression was unique to lung adenocarcinoma. Mechanistically, MK2 promotes nuclear translocation of caspase 3 leading to PARP1 cleavage and execution of cell death. While MK2 can directly phosphorylate caspase 3, neither phosphorylation status of caspase 3 nor the kinase activity of MK2 impacts caspase 3 activation, nuclear translocation and execution of cell death. Rather, a non-kinase function of MK2, specifically trafficking via its nuclear localization sequence, is required for caspase 3 mediated cell death. In summary this study highlights the importance of a non-enzymatic function of MK2 in the execution of apoptosis, which may be leveraged in the adjunctive treatment of NSCLC or other conditions where regulation of apoptosis is crucial.

cancer biology↗

bioRxiv2021

Rhodanese Rdl2 produces reactive sulfur species to scavenge hydroxyl radical and protect mitochondria

During aerobic respiration, mitochondria generate superoxide anion (O2*-), hydrogen peroxide (H2O2), and hydroxyl radical (HO*), and these reactive oxygen species (ROS) are detrimental to mitochondria. Mitochondrial damage is linked to a broad spectrum of pathologies such as Alzheimers disease, hemochromatosis, and diabetes. Mitochondria contain several enzymes for rapidly removing superoxide anion and hydrogen peroxide, but how they antagonize HO* is elusive, representing a loophole in the anti-ROS system. Herein, we discovered that Rhodanese 2 (Rdl2) is critical for maintaining the functionality and integrity of mitochondria under sub-lethal ROS stress in Saccharomyces cerevisiae. Rdl2 converts stable sulfur species (thiosulfate and dialkyl polysulfide) to reactive sulfane sulfur including persulfide that protects mitochondrial DNA via scavenging HO*. Surprisingly, hydrogen sulfide (H2S) promotes HO* production through stimulating the Fenton reaction, leading to increased DNA damage. Our study may reveal an ex-ante mean for antagonizing HO*, patching the loophole of the anti-ROS system in mitochondria.

microbiology↗

bioRxiv2021

The Hox gene Antennapedia regulates wing development through 20-hydroxyecdysone in insect

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species, Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila, and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20E. In turn, 20E signaling also up-regulates Antp. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28, and CPG9, essential for wing development. We propose, thus, that insect wing development occurs in an Antp-dependent manner.

developmental biology↗

bioRxiv2021

DNA methylation affects pre-mRNA transcriptional initiation and processing in Arabidopsis

BackgroundDNA methylation may regulate pre-mRNA transcriptional initiation and processing, thus affecting gene expression. Unlike animal cells, plants, especially Arabidopsis thaliana, have relatively low DNA methylation levels, limiting our ability to observe any correlation between DNA methylation and pre-mRNA processing using typical short-read sequencing. However, with newly developed long-read sequencing technologies, such as Oxford Nanopore Technology Direct RNA sequencing (ONT DRS), combined with whole-genome bisulfite sequencing, we were able to precisely analyze the relationship between DNA methylation and pre-mRNA transcriptional initiation and processing using DNA methylation-related mutants. ResultsUsing ONT DRS, we generated more than 2 million high-quality full-length long reads of native mRNA for each of the wild type Col-0 and mutants defective in DNA methylation, identifying a total of 117,474 isoforms. We found that low DNA methylation levels around splicing sites tended to prevent splicing events from occurring. The lengths of the poly(A) tail of mRNAs were positively correlated with DNA methylation. DNA methylation before transcription start sites or around transcription termination sites tended to result in gene-silencing or read-through events. Furthermore, using ONT DRS, we identified novel transcripts that we could not have otherwise, since transcripts with intron retention and fusion transcripts containing the uncut intergenic sequence tend not to be exported to the cytoplasm. Using the met1-3 mutant with activated constitutive heterochromatin regions, we confirmed the effects of DNA methylation on pre-mRNA processing. ConclusionThe combination of ONT DRS with whole-genome bisulfite sequencing was a powerful tool for studying the effects of DNA methylation on splicing site selection and pre-mRNA processing, and therefore regulation of gene expression.

plant biology↗

bioRxiv2021

Gallic Acid, a Methyl 3,4-Dihydroxybenzoate Derivative, Induces Neural Stem Cells to Differentiate and Proliferate

Differentiation and proliferation of neural stem cells (NSCs) are both important biological processes in cerebral neural network. However, these two capacities of NSCs are limited. Thus, the induction of differentiation and/or proliferation via administration of small molecules derived from natural plants can be considered as a potential approach to repair damaged neural networks. This paper reports that gallic acid, a catechol compound selected from derivatives of methyl 3,4-dihydroxybenzoate (MDHB), selectively induces NSCs to differentiate into immature neurons and promotes proliferation by activating phosphorylation of key proteins in the MAPK-ERK pathway. In addition, we found that 3,4-dihydroxybenzoic acid was the main active structure which could show neurotrophic activity. The substitution of carboxyl group on the benzene ring into ester group may promote differentiation on the basis of the structure of 3,4-dihydroxybenzoic acid. Meanwhile, the introduction of 5-hydroxyl group may promote proliferation. Generally, this study identified a natural catechol compound that promotes differentiation and proliferation of NSCs in vitro.

neuroscience↗

bioRxiv2020

Genome engineering of Nannochloropsis with large deletions for constructing microalgal minigenomes

Industrial microalgae are promising photosynthetic cell factories, yet tools for targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica we established a method to precisely and serially delete large genome fragments of ~100 kb from its 30.01-Mb nuclear genome. We started by identifying the "non-essential" chromosomal regions (i.e., low-expression region or LER) based on minimal gene expression under N-replete and N-depleted conditions. The largest such LER (LER1) is ~98 kb in size, located near the telomere of the 502.09 kb-long Chromosome 30 (Chr 30). We deleted 81 kb and further distal and proximal deletions of up to 110 kb (21.9% of Chr 30) in LER1 by dual targeting the boundaries with the episome-based CRISPR/Cas9 system. The telomere-deletion mutants showed normal telomeres consisting of CCCTAA repeats, revealing telomere regeneration capability after losing distal part of Chr 30. Interestingly, the deletions caused no significant alteration in growth, lipid production or photosynthesis (transcript-abundance change for < 3% genes under N depletion). We also performed double-deletion of both LER1 and LER2 (from Chr 9) that totals ~214 kb, and phenotypes are essentially normal. Therefore, loss of the large yet "non-essential" regions does not necessarily sacrifice important traits. Such serial targeted deletions of large genomic regions have not been reported in plants or microalgae, and will accelerate crafting minimal genomes as chassis for photosynthetic production.

biochemistry↗