The first successful observation of in-cell NMR signals of DNA and RNA in living human cells

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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Inhibition by selenium of DNA and RNA synthesis in normal and malignant human cells in vitro

Cancer Letters ◽

10.1016/0304-3835(92)90211-d ◽

1992 ◽

Vol 65 (1) ◽

pp. 43-49 ◽

Cited By ~ 14

Author(s):

Fikrat I. Abdullaev ◽

Christina MacVicar ◽

Gerald D. Frenkel

Keyword(s):

Rna Synthesis ◽

Human Cells ◽

Dna And Rna ◽

Dna And Rna Synthesis

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DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes

Nucleic Acids Research ◽

10.1093/nar/gkaa843 ◽

2020 ◽

Vol 48 (20) ◽

pp. e117-e117

Author(s):

Kun Tian ◽

Yongjian Guo ◽

Bingjie Zou ◽

Liang Wang ◽

Yun Zhang ◽

...

Keyword(s):

Rna Editing ◽

Human Cells ◽

Hairpin Dna ◽

Flap Endonuclease 1 ◽

Dna And Rna ◽

Rna Targets ◽

Single Base Mismatch ◽

Compact Size

Abstract Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

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Pores Formation in Porcine Sperm Incubated in Streptolysin O and Its Post Thawing Viability after Trehalose Treatment

Indian Journal of Animal Research ◽

10.18805/ijar.b-1302 ◽

2021 ◽

Author(s):

E. Jácome-Sosa ◽

M. Barrientos-Morales ◽

M.L. Juárez-Mosqueda ◽

B. Domínguez-Mancera

Keyword(s):

Pore Formation ◽

Membrane Integrity ◽

Control Group ◽

Dna And Rna ◽

Freezing Medium ◽

Streptolysin O ◽

Sperm Membrane ◽

Research Period ◽

Porcine Spermatozoa

Background: Streptolysin O (SLO), a pore-forming protein in plasma membrane (PM), has been used to internalize a variety of molecules (DNA and RNA) in cells. In sperm, however, SLO has only been used to release acrosomal contents. Its possible use as biotechnology in the cryopreservation of pig semen. However, porcine sperm are very sensitive to the freeze-thaw process. The study aimed to evaluate the pore formation in the PM, the addition of trehalose and the post cryopreservation viability of porcine spermatozoa using SLO.Methods: Research period was spring 2017- summer 2018. Thirty ejaculates from five mature boars were used. Semen was incubated in SLO 0.6 IU/ml and trehalose (added at 100, 200 and 400 ìM). Semen diluted in commercial diluent as control group. Presence of pores was checked by scanning electronic microscopy. To evaluate sperm membrane integrity and functional status the Coomassie stain with HOST test and the Chlortetracycline test were used. Result: It was found that SLO could form pores in the sperm cell membrane The addiction of 200 ìM trehalose to the freezing medium have different effects on the quality of boar sperm, showing highest motility and viability during the cooling process.

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A Chlamydia pneumoniae adhesin induces phosphatidylserine exposure on host cells

Nature Communications ◽

10.1038/s41467-019-12419-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Jan N. Galle ◽

Tim Fechtner ◽

Thorsten Eierhoff ◽

Winfried Römer ◽

Johannes H. Hegemann

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Pore Formation ◽

Chlamydia Pneumoniae ◽

Cytoplasmic Membrane ◽

Human Cells ◽

Host Cells ◽

Intracellular Pathogens ◽

Synthetic Membranes ◽

Human Pathogenic Bacterium

Abstract In mammalian cells, the internal and external leaflets of the plasma membrane (PM) possess different phospholipids. Phosphatidylserine (PS) is normally confined to the inner (cytoplasmic) membrane leaflet. Here we report that the adhesin CPn0473 of the human pathogenic bacterium Chlamydia pneumoniae (Cpn) binds to the PM of human cells and induces PS externalization but unexpectedly not apoptosis. PS externalization is increased in human cells exposed to infectious Cpn cells expressing increased CPn0473 and reduced in exposure to Cpn expressing decreased CPn0473. CPn0473 binds specifically to synthetic membranes carrying PS and stimulates pore formation. Asymmetric giant unilamellar vesicles (GUVs) in which PS is restricted to the inner leaflet reveal that CPn0473 induces PS externalization in the absence of other proteins. Thus our identification of CPn0473 as a bacterial PS translocator capable of specific and apoptosis-independent PS externalization during infection extends the spectrum of mechanisms intracellular pathogens use to enter host cells.

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High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140130 ◽

1995 ◽

Vol 53 ◽

pp. 752-753

Author(s):

B.A. Hamkalo ◽

S. Narayanswami ◽

A.P. Kausch

Keyword(s):

Nucleic Acid ◽

Interphase Nucleus ◽

Genome Mapping ◽

Precise Location ◽

Electron Microscope Level ◽

Rna Sequences ◽

Fundamental Interest ◽

Whole Cells ◽

Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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