Optimization of T4 phage engineering via CRISPR/Cas9

Abstract A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03–3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.

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Faculty Opinions recommendation of One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718007692.793477327 ◽

2013 ◽

Author(s):

Jakub Tolar ◽

Michael Vanden Oever

Keyword(s):

Genome Engineering ◽

Multiple Genes ◽

One Step ◽

Step Generation

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Freezing in Silicon at Large Undercooling

MRS Proceedings ◽

10.1557/proc-157-363 ◽

1989 ◽

Vol 157 ◽

Author(s):

P.A. Stolk ◽

A. Polman ◽

W.C. Sinke

Keyword(s):

Interface Temperature ◽

Limiting Factor ◽

Time Resolved ◽

Large Undercooling ◽

Explosive Crystallization ◽

Crystallization Speed ◽

Solid Liquid Interface ◽

Solid Liquid ◽

Rate Limiting

ABSTRACTPulsed laser irradiation is used to induce epitaxial explosive crystallization of amorphous silicon layers buried in a (100) oriented crystalline matrix. This process is mediated by a self-propagating liquid layer. Time-resolved determination of the crystallization speed combined with numerical calculation of the interface temperature shows that freezing in silicon saturates at 16 m/s for large undercooling (> 130 K). A comparison between data and different models for melting and freezing indicates that the crystallization behavior at large undercooling can be described correctly if the rate-limiting factor is assumed to be diffusion in liquid Si at the solid/liquid interface.

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Hormone-Induced Lipolysis and The Metabolic Pathways Providing Energy To The Cell: A Possible Role of Atp As a Rate-Limiting Factor

Lipids, Lipoproteins, and Drugs - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4684-3258-9_7 ◽

1975 ◽

pp. 105-122 ◽

Cited By ~ 1

Author(s):

G. Fassina ◽

P. Dorigo ◽

R. M. Gaion

Keyword(s):

Metabolic Pathways ◽

Limiting Factor ◽

Rate Limiting

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Recognized and Emerging Features of Erythropoietic and X-Linked Protoporphyria

Diagnostics ◽

10.3390/diagnostics12010151 ◽

2022 ◽

Vol 12 (1) ◽

pp. 151

Author(s):

Elena Di Pierro ◽

Francesca Granata ◽

Michele De Canio ◽

Mariateresa Rossi ◽

Andrea Ricci ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Aminolevulinic Acid ◽

Protoporphyrin Ix ◽

Limiting Factor ◽

Zinc Protoporphyrin ◽

Complete Understanding ◽

Inherited Disorders ◽

Rate Limiting ◽

Systemic Accumulation ◽

Made In

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inherited disorders resulting from defects in two different enzymes of the heme biosynthetic pathway, i.e., ferrochelatase (FECH) and delta-aminolevulinic acid synthase-2 (ALAS2), respectively. The ubiquitous FECH catalyzes the insertion of iron into the protoporphyrin ring to generate the final product, heme. After hemoglobinization, FECH can utilize other metals like zinc to bind the remainder of the protoporphyrin molecules, leading to the formation of zinc protoporphyrin. Therefore, FECH deficiency in EPP limits the formation of both heme and zinc protoporphyrin molecules. The erythroid-specific ALAS2 catalyses the synthesis of delta-aminolevulinic acid (ALA), from the union of glycine and succinyl-coenzyme A, in the first step of the pathway in the erythron. In XLP, ALAS2 activity increases, resulting in the amplified formation of ALA, and iron becomes the rate-limiting factor for heme synthesis in the erythroid tissue. Both EPP and XLP lead to the systemic accumulation of protoporphyrin IX (PPIX) in blood, erythrocytes, and tissues causing the major symptom of cutaneous photosensitivity and several other less recognized signs that need to be considered. Although significant advances have been made in our understanding of EPP and XLP in recent years, a complete understanding of the factors governing the variability in clinical expression and the severity (progression) of the disease remains elusive. The present review provides an overview of both well-established facts and the latest findings regarding these rare diseases.

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Neutrophil Extracellular Traps-DNase Balance and Autoimmunity

Cells ◽

10.3390/cells10102667 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2667

Author(s):

Andrea Angeletti ◽

Stefano Volpi ◽

Maurizio Bruschi ◽

Francesca Lugani ◽

Augusto Vaglio ◽

...

Keyword(s):

Lupus Erythematosus ◽

Causative Factor ◽

Neutrophil Extracellular Traps ◽

Limiting Factor ◽

Systemic Lupus ◽

Extracellular Traps ◽

Number Of Patients ◽

Selective Depletion ◽

Dna Antibodies ◽

Rate Limiting

Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNASE genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.

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Glypican-1 drives unconventional secretion of Fibroblast Growth Factor 2

10.1101/2021.11.11.468179 ◽

2021 ◽

Author(s):

Carola Sparn ◽

Eleni Dimou ◽

Annalena Meyer ◽

Roberto Saleppico ◽

Sabine Wegehingel ◽

...

Keyword(s):

Growth Factor ◽

Heparan Sulfate ◽

Fibroblast Growth Factor ◽

Fibroblast Growth Factor 2 ◽

Structural Basis ◽

Limiting Factor ◽

Fibroblast Growth ◽

Unconventional Secretion ◽

Rate Limiting

Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse sub-classes consisting of syndecans, perlecans, glypicans and others, Glypican-1 (GPC1) is both the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.

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Availability of Cosubstrates as a Rate-Limiting Factor

Drugs and Nutrients ◽

10.4324/9781003065128-6 ◽

2020 ◽

pp. 119-178

Author(s):

Gerard J. Mulder ◽

Klaas R. Krijgsheld

Keyword(s):

Limiting Factor ◽

Rate Limiting

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Dependence upon Bile Volume of the Biliary Excretion of Bromocresol Green and Amaranth in the Anaesthetized Rat

Australian Journal of Biological Sciences ◽

10.1071/bi9750339 ◽

1975 ◽

Vol 28 (4) ◽

pp. 339 ◽

Cited By ~ 1

Author(s):

Alan G Clark ◽

KarI M Rogers

Keyword(s):

Biliary Excretion ◽

Biliary Tree ◽

Bulk Flow ◽

Rate Equations ◽

Bromocresol Green ◽

Limiting Factor ◽

Cumulative Volume ◽

Rate Limiting ◽

Kinetics Of ◽

Anaesthetized Rat

The kinetics of the biliary excretion of both bromocresol green and amaranth are better described in terms of rate equations that are functions of the cumulative volume of bile excreted rather than of time. The rate of disappearance of bromocresol green from the liver also appears to depend on the volume of bile excreted rather than on time. It is proposed that bromocresol green, and probably also amaranth, rapidly equilibrates between the hepatic and biliary compartments as a result of reabsorption from the biliary tree and that the rate-limiting factor in the biliary excretion of these dyes is the removal of dye from the biliary tree by bulk flow.

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