Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP

CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum, yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing SpCas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum. Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.

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RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.00203-09 ◽

2009 ◽

Vol 9 (1) ◽

pp. 97-106 ◽

Cited By ~ 266

Author(s):

Eric R. Moellering ◽

Christoph Benning

Keyword(s):

Rna Interference ◽

Chlamydomonas Reinhardtii ◽

Droplet Size ◽

Lipid Droplet ◽

Lipid Droplets ◽

Biofuel Production ◽

Nitrogen Deprivation ◽

Green Algal ◽

Lipid Droplet Size ◽

Lipid Droplet Protein

ABSTRACT Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.

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Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

10.1101/2020.12.09.418624 ◽

2020 ◽

Author(s):

Wei Wang ◽

Bin Tian ◽

Qianli Pan ◽

Yueying Chen ◽

Fei He ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Genome Editing ◽

Agronomic Traits ◽

Wheat Genome ◽

Editing Efficiency ◽

Francisella Novicida ◽

Guide Rna ◽

Guide Rnas ◽

Protospacer Adjacent Motif ◽

Endogenous Genes

AbstractThe development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

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AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

Nature Communications ◽

10.1038/s41467-021-24017-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Liyang Zhang ◽

John A. Zuris ◽

Ramya Viswanathan ◽

Jasmine N. Edelstein ◽

Rolf Turk ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Genome Editing ◽

Nk Cell ◽

Basic Research ◽

Cell Recognition ◽

Site Specific ◽

Editing Efficiency ◽

Rapid Generation ◽

Therapeutic Cell

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

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Indolylbenzothiadiazoles as highly tunable fluorophores for imaging lipid droplet accumulation in astrocytes and glioblastoma cells

RSC Advances ◽

10.1039/d1ra04419b ◽

2021 ◽

Vol 11 (39) ◽

pp. 23960-23967

Author(s):

Kilian Colas ◽

Karl O. Holmberg ◽

Linus Chiang ◽

Susanne Doloczki ◽

Fredrik J. Swartling ◽

...

Keyword(s):

Lipid Droplet ◽

Lipid Droplets ◽

Glioblastoma Cells ◽

Photophysical Study

We present an extensive photophysical study of a series of fluorescent indolylbenzothiadiazole derivatives and their ability to specifically image lipid droplets in astrocytes and glioblastoma cells.

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Identification of Perilipin-2 as a lipid droplet protein regulated in oocytes during maturation

Reproduction Fertility and Development ◽

10.1071/rd10091 ◽

2010 ◽

Vol 22 (8) ◽

pp. 1262 ◽

Cited By ~ 35

Author(s):

Xing Yang ◽

Kylie R. Dunning ◽

Linda L.-Y. Wu ◽

Theresa E. Hickey ◽

Robert J. Norman ◽

...

Keyword(s):

Lipid Droplet ◽

Lipid Content ◽

Lipid Droplets ◽

Intracellular Lipids ◽

Epidermal Growth ◽

Novel Method ◽

Perilipin 2 ◽

Lipid Droplet Protein

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus–oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins Perilipin, Perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only Perilipin-2 was associated with lipid droplets in the oocyte. In COCs, Perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce Perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that Perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, Perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

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Premature termination by human RNA polymerase II occurs temporally in the adenovirus major late transcriptional unit

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2031-2040.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2031-2040

Author(s):

M Mok ◽

A Maderious ◽

S Chen-Kiang

Keyword(s):

Dna Replication ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Premature Termination ◽

Adenovirus Type ◽

Transcriptional Unit ◽

Late Gene ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Adenovirus Type 5

We have recently demonstrated pausing and premature termination of transcription by eucaryotic RNA polymerase II at specific sites in the major late transcriptional unit of adenovirus type 2 in vivo and in vitro. In further developing this as a system for studying eucaryotic termination control, we found that prematurely terminated transcripts of 175 and 120 nucleotides also occur in adenovirus type 5-infected cells. In both cases, premature termination occurs temporally, being found only during late times of infection, not at early times before DNA replication or immediately after the onset of DNA replication when late gene expression has begun (intermediate times). To examine the phenomenon of premature termination further, a temperature-sensitive mutant virus, adenovirus type 5 ts107, was used to uncouple DNA replication and transcription. DNA replication is defective in this mutant at restrictive temperatures. We found that premature termination is inducible at intermediate times by shifting from a permissive temperature to a restrictive temperature, allowing continuous transcription in the absence of continuous DNA replication. No premature termination occurs when the temperature is shifted up at early times before DNA replication. Our data suggest that premature termination of transcription is dependent on both prior synthesis of new templates and cumulative late gene transcription but does not require continuous DNA replication.

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Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx.

Genetics ◽

10.1093/genetics/131.4.895 ◽

1992 ◽

Vol 131 (4) ◽

pp. 895-903

Author(s):

M A Mortin ◽

R Zuerner ◽

S Berger ◽

B J Hamilton

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Thoracic Segment ◽

Mutant Phenotype ◽

Analogous Structure ◽

Gene Encoding ◽

Recessive Lethal ◽

The Third ◽

Complementation Groups

Abstract Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of a structure of the third thoracic segment, the capitellum, into the analogous structure of the second thoracic segment, the wing. This mutant phenotype is also caused by genetically reducing the cellular concentration of the transcription factor Ultrabithorax (Ubx). To recover mutations in the 140,000-D second-largest subunit of RNA polymerase II (RpII140) and determine whether any can cause a mutant phenotype similar to Ubx we attempted to identify all recessive-lethal mutable loci in a 340-kilobase deletion including this and other loci. One of the 13 complementation groups in this region encodes RpII140. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of Ubx protein is reduced by mutations in Ubx.

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HSD17B13: A Potential Therapeutic Target for NAFLD

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.824776 ◽

2022 ◽

Vol 8 ◽

Author(s):

Hai-bo Zhang ◽

Wen Su ◽

Hu Xu ◽

Xiao-yan Zhang ◽

You-fei Guan

Keyword(s):

Liver Disease ◽

Lipid Droplet ◽

Lipid Droplets ◽

Critical Role ◽

Chronic Liver ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Nonalcoholic Fatty Liver ◽

Promising Target ◽

Associated Proteins

Nonalcoholic fatty liver disease (NAFLD), especially in its inflammatory form (steatohepatitis, NASH), is closely related to the pathogenesis of chronic liver disease. Despite substantial advances in the management of NAFLD/NASH in recent years, there are currently no efficacious therapies for its treatment. The biogenesis and expansion of lipid droplets (LDs) are critical pathophysiological processes in the development of NAFLD/NASH. In the past decade, increasing evidence has demonstrated that lipid droplet-associated proteins may represent potential therapeutic targets for the treatment of NAFLD/NASH given the critical role they play in regulating the biogenesis and metabolism of lipid droplets. Recently, HSD17B13, a newly identified liver-enriched, hepatocyte-specific, lipid droplet-associated protein, has been reported to be strongly associated with the development and progression of NAFLD/NASH in both mice and humans. Notably, human genetic studies have repeatedly reported a robust association of HSD17B13 single nucleotide polymorphisms (SNPs) with the occurrence and severity of NAFLD/NASH and other chronic liver diseases (CLDs). Here we briefly overview the discovery, tissue distribution, and subcellular localization of HSD17B13 and highlight its important role in promoting the pathogenesis of NAFLD/NASH in both experimental animal models and patients. We also discuss the potential of HSD17B13 as a promising target for the development of novel therapeutic agents for NAFLD/NASH.

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