scholarly journals Improved architectures for flexible DNA production using retrons across kingdoms of life

2021 ◽  
Author(s):  
Santiago C Lopez ◽  
Kate D Crawford ◽  
Santi Bhattarai-Kline ◽  
Seth L Shipman
Keyword(s):  
Genome Editing ◽  
Eukaryotic Cells ◽  
Target Cells ◽  
Exogenous Dna ◽  
Reverse Transcriptases ◽  
Non Coding Rna ◽  
Dna Production ◽  
Cultured Human Cells ◽  
Template Dna

Exogenous DNA is a critical molecular tool for biology. This is particularly true for gene editing, where exogenous DNA can be used as a template to introduce precise changes to the sequence of a cell's genome. This DNA is typically synthesized or assembled in vitro and then delivered to target cells. However, delivery can be inefficient, and low abundance of template DNA may be one reason that precise editing typically occurs at a low rate. It has recently been shown that producing DNA inside cells can using reverse transcriptases can increase the efficiency of genome editing. One tool to produce that DNA is a retron, a bacterial retroelement that has an endogenous role in phage defense. However, little effort has been directed at optimizing the retron for production of designed sequences when used as a component of biotechnology. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. We also test architectures of the retron operon that enable efficient reverse transcription across kingdoms of life from bacteria, to yeast, to cultured human cells. We find that gains in DNA production using modified retrons are portable from prokaryotic to eukaryotic cells. Finally, we demonstrate that increased production of RT-DNA results in more efficient genome editing in both prokaryotic and eukaryotic cells. These experiments provide a general framework for production of DNA using a retron for biotechnological applications.

scholarly journals Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

2019 ◽  
Author(s):  
Jes Alexander ◽  
Gregory M. Findlay ◽  
Martin Kircher ◽  
Jay Shendure
Keyword(s):  
Genome Editing ◽  
Cpg Island ◽  
Exogenous Dna ◽  
Cpg Dinucleotides ◽  
Epigenome Editing ◽  
Functional Consequences ◽  
Cpg Dinucleotide ◽  
Methylation Patterns

AbstractRecent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double stranded break (DSB), followed by NHEJ or HDR mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, i.e. to drive replacement of the DNA sequence intervening the dual cuts via NHEJ. In a proof-of-concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event, and opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

scholarly journals Competition between the Yops of Yersinia enterocolitica for Delivery into Eukaryotic Cells: Role of the SycE Chaperone Binding Domain of YopE

2000 ◽  
Vol 182 (17) ◽  
pp. 4811-4821 ◽  
Author(s):  
Aoife P. Boyd ◽  
Isabelle Lambermont ◽  
Guy R. Cornelis
Keyword(s):  
Binding Site ◽  
In Vitro Release ◽  
Inhibitory Effect ◽  
Binding Domain ◽  
Eukaryotic Cells ◽  
Target Cells ◽  
Wild Type ◽  
Animal Cells

ABSTRACT A type III secretion-translocation system allowsYersinia adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca2+-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant Yersinia devoid of most of the Yop effectors (ΔHOPEM and ΔTHE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE15-Cya as efficiently as YopE130-Cya. They were also able to deliver YopEΔ17–77. SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type Yersinia suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca2+ chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.

scholarly journals 5' modifications improve potency and efficacy of DNA donors for precision genome editing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Krishna S Ghanta ◽  
Zexiang Chen ◽  
Aamir Mir ◽  
Gregoriy A Dokshin ◽  
Pranathi M Krishnamurthy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Triethylene Glycol ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Cultured Human Cells ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

scholarly journals Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector

2019 ◽  
Author(s):  
Iara Rocchi ◽  
Charles Ericson ◽  
Kyle E. Malter ◽  
Sahar Zargar ◽  
Fabian Eisenstein ◽  
...  
Keyword(s):  
Fall Armyworm ◽  
Nuclease Activity ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Target Cells ◽  
Sf9 Cells ◽  
Murine Macrophage ◽  
Related Proteins ◽  
Novel Delivery Systems

ABSTRACTMany bacteria interact with target organisms using syringe-like structures called Contractile Injection Systems (CIS). CIS structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. Recent evidence shows that CIS are specialized to puncture membranes and often deliver effectors to target cells. In many cases, CIS mediate trans-kingdom interactions between bacteria and eukaryotes, however the effectors delivered to target cells and their mode of action are often unknown. In this work, we establish an in vitro model to study a CIS called Metamorphosis Associated Contractile structures (MACs) that target eukaryotic cells. We show that MACs kill two eukaryotic cell lines, Fall Armyworm Sf9 cells and J774A.1 murine macrophage cells through the action of a newly identified MAC effector, termed Pne1. To our knowledge, Pne1 is the first CIS effector exhibiting nuclease activity against eukaryotic cells. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a first step toward understanding structures with the potential to be developed as novel delivery systems for eukaryotic hosts.

scholarly journals Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Jes Alexander ◽  
Gregory M. Findlay ◽  
Martin Kircher ◽  
Jay Shendure
Keyword(s):  
Genome Editing ◽  
Cpg Island ◽  
Strand Break ◽  
End Joining ◽  
Exogenous Dna ◽  
Cpg Dinucleotides ◽  
Epigenome Editing ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

PEG-Glu-PEI-Dexamethasone Conjugates: Synthesis, Characterization and In Vitro Gene Transfer Properties

2013 ◽  
Vol 747 ◽  
pp. 147-147
Author(s):  
Tae Hun Kim ◽  
Hye Choi ◽  
Gwang Sig Yu ◽  
Joon Sig Choi
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Serum Proteins ◽  
Target Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Surface Charges ◽  
Exogenous Dna ◽  
Cellular Expression

Gene therapy has the ability to treat diseases by delivering exogenous DNA into the nuclei of target cells to express a therapeutic protein. In intravenous gene delivery, free oligonucleotides and DNA are rapidly degraded by serum nucleases in the blood before they can reach the target site. Therefore, the main goal for gene therapy is not just to obtain cellular expression of an exogenous gene per se, but also to develop non-toxic and efficient carriers. Because of its abundant positive surface charges, PEI is able to condense the negatively charged DNA into PEI/DNA complexes with a net positive charge. PEG facilitates the formation of polyplexes with improved solubility, reduced aggregation, lower cytotoxicity, and possibly decreases opsonization with serum proteins in the bloodstream. Also, dexamethasone is the potent ligand of the glucocorticoid receptor which facilitates the transfer into nucleus, and it is known to enlarge the nuclear pore complexes. In this study, PEG-Glu-PEI-Dexa was synthesized as a kind of biodegradable polycation for gene delivery.

scholarly journals BACTENECINS AS CELL-PENETRATING PEPTIDES

2019 ◽  
Vol 19 (1S) ◽  
pp. 178-179
Author(s):  
O V Shamova ◽  
A S Nazarov ◽  
P M Kopeykin ◽  
I V Kudryavtsev ◽  
N A Grudinina ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Eukaryotic Cells ◽  
Target Cells ◽  
Medium Temperature ◽  
Cell Penetrating ◽  
Internalization Process ◽  
Infected Cells ◽  
Truncated Variants

Cell-Penetrating Peptides (CPPs) are molecules that can easily internalize into eukaryotic cells, as well as deliver across their membranes a variety of compounds (proteins, nucleic acids, liposomes, nanoparticles, etc.). CPPs are considered as promising components of anticancer drugs, serving for delivery of active ingredients into malignant cells, therefore, a detailed study of a mechanism of action of CPPs and search for novel, more effective peptides are vital tasks of current biological and medical research. An ability of proline-rich peptides bactenecins (ChBac5, ChBac3.4, mini-ChBac7.5Na) and their truncated variants to penetrate into eukaryotic cells has been explored. By means of flow cytometry and confocal microscopy, we found that these peptides, tagged with a fluorescent dye BODIPY FL, rapidly penetrated into tumor cells and, to a lesser extent, into normal mammalian cells in vitro. The dependence of the internalization process on the medium temperature and energy metabolism of target cells was studied. The obtained data on the cell-penetratin activity of caprine bactenecins confirm the prospect of further investigations of these peptides as prototypes of new compounds - carriers of drugs into malignant or infected cells.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

The Hierarchic Order of Intermediate Filament Structure and Assembly

1985 ◽  
Vol 43 ◽  
pp. 722-725
Author(s):  
U. Aebi ◽  
E.C. Glavaris ◽  
R. Eichner
Keyword(s):  
Tissue Specificity ◽  
Structural Model ◽  
Eukaryotic Cells ◽  
Cdna Sequencing ◽  
Filament Structure ◽  
Keratin Filaments ◽  
Isoelectric Points ◽  
Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

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