scholarly journals Unbiased identification of nanoparticle cell uptake mechanism via a genome-wide CRISPR/Cas9 knockout screen

2020 ◽  
Author(s):  
Eric L. Van Nostrand ◽  
Sarah A. Barnhill ◽  
Alexander A. Shishkin ◽  
David A. Nelles ◽  
Eric Byeon ◽  
...  
Keyword(s):  
Cell Uptake ◽  
Target Cells ◽  
Uptake Mechanism ◽  
Therapeutic Delivery ◽  
Genome Wide ◽  
A Genome ◽  
Technical Reproducibility ◽  
Genome Screening ◽  
Major Bottleneck ◽  
Target Effects

AbstractA major bottleneck in nanocarrier and macromolecule development for therapeutic delivery is our limited understanding of the processes involved in their uptake into target cells. This includes their active interactions with membrane transporters that co-ordinate cellular uptake and processing. Current strategies to elucidate the mechanism of uptake, such as painstaking manipulation of individual effectors with pharmacological inhibitors or specific genetic knockdowns, are limited in scope and biased towards previously studied pathways or the intuition of the investigators. Furthermore, each of these approaches present significant off-target effects, clouding the outcomes. We set out to develop and examine an unbiased whole-genome screening approach using pooled CRISPR/Cas9 libraries for its ability to provide a robust and rapid approach to identify novel effectors of material uptake. Enabling this, we developed a methodology termed fast-library of inserts (FLI)-seq for library preparation and quantitative readout of pooled screens that shows improved technical reproducibility and is easier to perform than existing methods. In this proof-of-concept study we use FLI-seq to identify a solute carrier protein family member, SLC18B1, as a transporter for polymeric micellar nanoparticles, confirming the viability for this approach to yield novel insights into uptake mechanisms.

scholarly journals Whole-genome screening identifies proteins localized to distinct nuclear bodies

2013 ◽  
Vol 203 (1) ◽  
pp. 149-164 ◽  
Author(s):  
Ka-wing Fong ◽  
Yujing Li ◽  
Wenqi Wang ◽  
Wenbin Ma ◽  
Kunpeng Li ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Nuclear Bodies ◽  
Cajal Bodies ◽  
Nuclear Speckles ◽  
Genome Wide ◽  
A Genome ◽  
Human Proteins ◽  
Genome Screening ◽  
Interchromatin Space ◽  
Polycomb Bodies

The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.

scholarly journals Cardiomyocyte uptake mechanism of a hydroxyapatite nanoparticle mediated gene delivery system

2020 ◽  
Vol 11 ◽  
pp. 1685-1692
Author(s):  
Hiroaki Komuro ◽  
Masahiro Yamazoe ◽  
Kosuke Nozaki ◽  
Akiko Nagai ◽  
Tetsuo Sasano
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Cell Uptake ◽  
Viral Gene ◽  
Target Cells ◽  
Gene Delivery System ◽  
Uptake Mechanism ◽  
Delivery Vector ◽  
Low Cytotoxicity ◽  
Viral Gene Delivery

Gene therapy has been explored as a future alternative for treating heart disease. Among several gene delivery systems aimed at penetrating specific target cells, we focused on safe and non-viral gene delivery materials with a high transfection efficiency. Although various techniques have been developed, the mechanisms underlying the cellular uptake of gene delivery materials have not yet been sufficiently studied in cardiomyocytes. The aim of this study was to determine how hydroxyapatite (HAp) nanoparticles contribute to the delivery of plasmid DNA (pDNA) into cardiomyocytes. We fabricated HAp nanoparticles using the water-in-oil (W/O) emulsion method and used these nanoparticles as the delivery vector for transfecting cardiomyocyte-derived HL-1 cells. HAp exhibited particles on the nanoscale and with a low cytotoxicity in HL-1 cells. The transfection assay performed with several endocytosis inhibitors suggested that the HAp/pDNA complexes were internalized by HL-1 cells through macropinocytosis. Furthermore, this HL-1 cell uptake was generated in response to HAp stimulation. Thus, HAp is a positive regulator of macropinocytosis in HL-1 cells and a good system for gene delivery in cardiomyocytes.

scholarly journals Incomplete MyoD-induced transdifferentiation is associated with chromatin remodeling deficiencies

2017 ◽  
Author(s):  
Dinesh Manandhar ◽  
Lingyun Song ◽  
Ami Kabadi ◽  
Jennifer Kwon ◽  
Lee Edsall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
Global Gene Expression ◽  
Chromatin Accessibility ◽  
Marker Genes ◽  
Target Cells ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Quantitative Analyses

Our current understanding of cellular transdifferentiation systems is limited. It is oftentimes unknown, at a genome-wide scale, how much transdifferentiated cells differ quantitatively from both the starting cells and the target cells. Focusing on transdifferentiation of primary human skin fibroblasts by forced expression of myogenic transcription factor MyoD, we performed quantitative analyses of gene expression and chromatin accessibility profiles of transdifferentiated cells compared to fibroblasts and myoblasts. In this system, we find that while many of the early muscle marker genes are reprogrammed, global gene expression and accessibility changes are still incomplete when compared to myoblasts. In addition, we find evidence of epigenetic memory in the transdifferentiated cells, with reminiscent features of fibroblasts being visible both in chromatin accessibility and gene expression. Quantitative analyses revealed a continuum of changes in chromatin accessibility induced by MyoD, and a strong correlation between chromatin-remodeling deficiencies and incomplete gene expression reprogramming. Classification analyses identified genetic and epigenetic features that distinguish reprogrammed from non-reprogrammed sites, and suggested ways to potentially improve transdifferentiation efficiency. Our approach for combining gene expression, DNA accessibility, and protein-DNA binding data to quantify and characterize the efficiency of cellular transdifferentiation on a genome-wide scale can be applied to any transdifferentiation system.

scholarly journals A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

2020 ◽  
Author(s):  
Bonaventure Boris ◽  
Rebendenne Antoine ◽  
Garcia de Gracia Francisco ◽  
McKellar Joe ◽  
Tauziet Marine ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Helicase ◽  
Target Cells ◽  
Knock Out ◽  
Ribonucleoprotein Complexes ◽  
Genome Wide ◽  
A Genome ◽  
Positive Strand Rna ◽  
Antiviral Inhibitor ◽  
Hiv 1

AbstractGenome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. Here, we took advantage of the ability of interferon (IFN) to restrict HIV-1 infection, in order to create an environment hostile to replication and reveal new inhibitors through a CRISPR screen. This approach led to the identification of the RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 increased HIV-1 DNA accumulation and infection in cell lines and primary cells, irrespectively of IFN treatment. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant of DDX42 increased infection. Importantly, DDX42 impacted retrotransposition of long interspersed elements-1 (LINE-1), infection with other retroviruses and positive-strand RNA viruses, including Chikungunya virus (CHIKV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, DDX42 did not inhibit infection with negative-strand RNA viruses such as influenza A virus (IAV), arguing against a general, unspecific effect on target cells. Proximity ligation assays showed DDX42 in the vicinity of viral elements during infection, and RNA immunoprecipitation confirmed DDX42 interaction with LINE-1 RNAs. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results identify DDX42 as a new, broadly active intrinsic antiviral inhibitor.

scholarly journals Targeting Cancer Cells Overexpressing Folate Receptors with New Terpolymer-Based Nanocapsules: Toward a Novel Targeted DNA Delivery System for Cancer Therapy

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1275
Author(s):  
Elena Bellotti ◽  
Maria Grazia Cascone ◽  
Niccoletta Barbani ◽  
Daniela Rossin ◽  
Raffaella Rastaldo ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Cancer Cells ◽  
Targeted Delivery ◽  
Genetic Material ◽  
Cell Uptake ◽  
Target Cells ◽  
Folate Receptors ◽  
Good Ability ◽  
Wide Range ◽  
Target Effects

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with folic acid to actively target the folate receptors overexpressed on a variety of cancer cells. The nanocapsules demonstrate a good ability of encapsulating and releasing DNA. Moreover, the presence of the targeting moieties on the surface of the nanocapsules favors cell uptake, opening up the possibility of more effective therapies.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

A genome-wide linkage scan for familial partial lipodystrophy susceptibility genes in a German kindreds

2007 ◽  
Vol 30 (4) ◽  
pp. 86
Author(s):  
M. Lanktree ◽  
J. Robinson ◽  
J. Creider ◽  
H. Cao ◽  
D. Carter ◽  
...  
Keyword(s):  
Subcutaneous Fat ◽  
Visceral Obesity ◽  
Fat Distribution ◽  
Dominant Negative ◽  
Molecular Forms ◽  
Partial Lipodystrophy ◽  
Genome Wide ◽  
A Genome ◽  
Familial Partial Lipodystrophy ◽  
Promoter Mutations

Background: In Dunnigan-type familial partial lipodystrophy (FPLD) patients are born with normal fat distribution, but subcutaneous fat from extremities and gluteal regions are lost during puberty. The abnormal fat distribution leads to the development of metabolic syndrome (MetS), a cluster of phenotypes including hyperglycemia, dyslipidemia, hypertension, and visceral obesity. The study of FPLD as a monogenic model of MetS may uncover genetic risk factors of the common MetS which affects ~30% of adult North Americans. Two molecular forms of FPLD have been identified including FPLD2, resulting from heterozygous mutations in the LMNA gene, and FPLD3, resulting from both heterozygous dominant negative and haploinsufficiency mutations in the PPARG gene. However, many patients with clinically diagnosed FPLD have no mutation in either LMNA or PPARG, suggesting the involvement of additional genes in FPLD etiology. Methods: Here, we report the results of an Affymetrix 10K GeneChip microarray genome-wide linkage analysis study of a German kindred displaying the FPLD phenotype and no known lipodystrophy-causing mutations. Results: The investigation identified three chromosomal loci, namely 1q, 3p, and 9q, with non-parametric logarithm of odds (NPL) scores >2.7. While not meeting the criteria for genome-wide significance, it is interesting to note that the 1q and 3p peaks contain the LMNA and PPARG genes respectively. Conclusions: Three possible conclusions can be drawn from these results: 1) the peaks identified are spurious findings, 2) additional genes physically close to LMNA, PPARG, or within 9q, are involved in FPLD etiology, or 3) alternative disease causing mechanisms not identified by standard exon sequencing approaches, such as promoter mutations, alternative splicing, or epigenetics, are also responsible for FPLD.

Sign in / Sign up

Export Citation Format

Share Document