Viruses as vectors for the delivery of gene-editing reagents

A significant challenge for plant gene editing is the delivery of editing reagents to germline or regenerable cells to recover heritable genetic modifications. Reagent delivery using biolistics or Agrobacterium is only possible with a limited range of species and genotypes, and inefficient editing or lengthy tissue culture steps further limit throughput. Viruses are natural vectors for nucleic acids, and both DNA and RNA plant viruses have been engineered to extend or replace conventional vectors for delivery of gene editing reagents. Here, we review aspects of viral biology essential for engineering vectors, highlight landmark studies using viruses to overcome traditional limitations in gene editing, and outline important considerations for the use of viral vectors in new systems or for new targets. Motivated by fundamental differences in both their infection modes and utility as vectors, DNA and RNA viruses are treated separately.

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A LABILE INTRANUCLEAR RNA ASSOCIATED WITH THE DEVELOPMENT OF ADENOVIRUSES

Journal of Experimental Medicine ◽

10.1084/jem.119.3.433 ◽

1964 ◽

Vol 119 (3) ◽

pp. 433-442 ◽

Cited By ~ 16

Author(s):

Heather Donald Mayor

Keyword(s):

Nucleic Acids ◽

Acridine Orange ◽

Viral Protein ◽

Rna Viruses ◽

Monkey Kidney ◽

Kidney Cells ◽

Viral Dna ◽

Dna And Rna ◽

Double Stranded Dna ◽

Cellular Dna

The nucleic acids produced intracellularly during the replication cycles of both DNA and RNA viruses can now be identified rapidly using a sensitized procedure based on staining with the fluorochrome acridine orange. Cellular DNA, viral DNA (both single and double stranded forms), cellular RNA, and RNA arising as a result of viral stimulus can be differentiated. The intracellular development of virus specific DNA, RNA, and protein has been studied in monkey kidney cells infected with adenoviruses types 3 and 7. It has been possible to detect a labile RNA in the nucleus from 16 to 20 hours after inoculation. When the cultures are treated with puromycin at this time, this RNA can be accumulated under certain conditions in the nucleus and demonstrated cytochemically. At the same time the production of specific viral protein as determined by staining with fluorescein-labeled antibodies is markedly inhibited. However, intranuclear double stranded DNA continues to be formed for a time. When puromycin is added to the system early in the eclipse period virus-specific DNA and labile RNA cannot be detected.

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Comparison of DNA and RNA sequencing of total nucleic acids from human cervix for metagenomics

Scientific Reports ◽

10.1038/s41598-021-98452-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Laila Sara Arroyo Mühr ◽

Joakim Dillner ◽

Agustin Enrique Ure ◽

Karin Sundström ◽

Emilie Hultin

Keyword(s):

Nucleic Acids ◽

Dna Sequencing ◽

Rna Sequencing ◽

Reverse Transcription ◽

Rna Viruses ◽

Dna And Rna ◽

Dnase Treatment ◽

Similarities And Differences ◽

Viral Sequences ◽

Human Cervix

AbstractAlthough metagenomics and metatranscriptomics are commonly used to identify bacteria and viruses in human samples, few studies directly compare these strategies. We wished to compare DNA and RNA sequencing of bacterial and viral metagenomes and metatranscriptomes in the human cervix. Total nucleic acids from six human cervical samples were subjected to DNA and RNA sequencing. The effect of DNase-treatment before reverse transcription to cDNA were also analyzed. Similarities and differences in the metagenomic findings with the three different sequencing approaches were evaluated. A higher proportion of human sequences were detected by DNA sequencing (93%) compared to RNA sequencing without (76%) and with prior DNase-treatment (11%). On the contrary, bacterial sequences increased 17 and 91 times. However, the number of detected bacterial genera were less by RNA sequencing, suggesting that only a few contribute to most of the bacterial transcripts. The viral sequences were less by RNA sequencing, still twice as many virus genera were detected, including some RNA viruses that were missed by DNA sequencing. Metatranscriptomics of total cDNA provided improved detection of mainly transcribed bacteria and viruses in cervical swabs as well as detection of RNA viruses, compared to metagenomics.

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Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA–CRISPR/Cas12a Assay

Frontiers in Microbiology ◽

10.3389/fmicb.2020.610872 ◽

2020 ◽

Vol 11 ◽

Author(s):

Rashid Aman ◽

Ahmed Mahas ◽

Tin Marsic ◽

Norhan Hassan ◽

Magdy M. Mahfouz

Keyword(s):

Pathogen Detection ◽

Rna Viruses ◽

Plant Viruses ◽

Nucleic Acid Amplification ◽

High Rate ◽

Detection Methods ◽

One Pot ◽

Robust Detection ◽

Dna And Rna ◽

Single Temperature

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

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Graphene Oxide: A Smart (Starting) Material for Natural Methylxanthines Adsorption and Detection

Molecules ◽

10.3390/molecules24234247 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4247 ◽

Cited By ~ 5

Author(s):

Rita Petrucci ◽

Isabella Chiarotto ◽

Leonardo Mattiello ◽

Daniele Passeri ◽

Marco Rossi ◽

...

Keyword(s):

Electrical Conductivity ◽

Graphene Oxide ◽

Nucleic Acids ◽

Reduced Graphene Oxide ◽

Electrochemical Sensors ◽

Enzymatic Digestion ◽

Biologically Active ◽

Polar Solvents ◽

Dna And Rna ◽

Reduced Graphene

Natural methylxanthines, caffeine, theophylline and theobromine, are widespread biologically active alkaloids in human nutrition, found mainly in beverages (coffee, tea, cocoa, energy drinks, etc.). Their detection is thus of extreme importance, and many studies are devoted to this topic. During the last decade, graphene oxide (GO) and reduced graphene oxide (RGO) gained popularity as constituents of sensors (chemical, electrochemical and biosensors) for methylxanthines. The main advantages of GO and RGO with respect to graphene are the easiness and cheapness of synthesis, the notable higher solubility in polar solvents (water, among others), and the higher reactivity towards these targets (mainly due to – interactions); one of the main disadvantages is the lower electrical conductivity, especially when using them in electrochemical sensors. Nonetheless, their use in sensors is becoming more and more common, with the obtainment of very good results in terms of selectivity and sensitivity (up to 5.4 × 10−10 mol L−1 and 1.8 × 10−9 mol L−1 for caffeine and theophylline, respectively). Moreover, the ability of GO to protect DNA and RNA from enzymatic digestion renders it one of the best candidates for biosensors based on these nucleic acids. This is an up-to-date review of the use of GO and RGO in sensors.

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Triazole-Modified Nucleic Acids for the Application in Bioorganic and Medicinal Chemistry

Biomedicines ◽

10.3390/biomedicines9060628 ◽

2021 ◽

Vol 9 (6) ◽

pp. 628

Author(s):

Dagmara Baraniak ◽

Jerzy Boryski

Keyword(s):

Nucleic Acids ◽

State Of The Art ◽

Modified Oligonucleotides ◽

Synthetic Genes ◽

Chemically Modified ◽

Amide Linkage ◽

Dna And Rna ◽

Modified Dna ◽

Modified Nucleic Acids ◽

Non Coding Rnas

This review covers studies which exploit triazole-modified nucleic acids in the range of chemistry and biology to medicine. The 1,2,3-triazole unit, which is obtained via click chemistry approach, shows valuable and unique properties. For example, it does not occur in nature, constitutes an additional pharmacophore with attractive properties being resistant to hydrolysis and other reactions at physiological pH, exhibits biological activity (i.e., antibacterial, antitumor, and antiviral), and can be considered as a rigid mimetic of amide linkage. Herein, it is presented a whole area of useful artificial compounds, from the clickable monomers and dimers to modified oligonucleotides, in the field of nucleic acids sciences. Such modifications of internucleotide linkages are designed to increase the hybridization binding affinity toward native DNA or RNA, to enhance resistance to nucleases, and to improve ability to penetrate cell membranes. The insertion of an artificial backbone is used for understanding effects of chemically modified oligonucleotides, and their potential usefulness in therapeutic applications. We describe the state-of-the-art knowledge on their implications for synthetic genes and other large modified DNA and RNA constructs including non-coding RNAs.

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Lactoferrin affects rhinovirus B-14 entry into H1-HeLa cells

Archives of Virology ◽

10.1007/s00705-021-04993-4 ◽

2021 ◽

Vol 166 (4) ◽

pp. 1203-1211

Author(s):

Caio Bidueira Denani ◽

Antonio Real-Hohn ◽

Carlos Alberto Marques de Carvalho ◽

Andre Marco de Oliveira Gomes ◽

Rafael Braga Gonçalves

Keyword(s):

Innate Immune System ◽

Rna Viruses ◽

Innate Immune ◽

Bovine Lactoferrin ◽

Respiratory Illnesses ◽

Dna And Rna ◽

Viral Particles ◽

The Common ◽

Additional Influence

AbstractLactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

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Characterization of nucleic acids from extracellular vesicle-enriched human sweat

BMC Genomics ◽

10.1186/s12864-021-07733-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Geneviève Bart ◽

Daniel Fischer ◽

Anatoliy Samoylenko ◽

Artem Zhyvolozhnyi ◽

Pavlo Stehantsev ◽

...

Keyword(s):

Nucleic Acids ◽

Human Genome ◽

Body Fluids ◽

Lower Percentage ◽

Rna Seq ◽

Protein Coding ◽

Human Sweat ◽

Dna And Rna ◽

Ribonucleoprotein Complexes ◽

Eccrine Glands

Abstract Background The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. Results We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7–45%), with 50–60% of those reads mapping to unannotated region of the genome and 30–55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. Conclusions Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

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VirusTaxo: Taxonomic classification of virus genome using multi-class hierarchical classification by k-mer enrichment

10.1101/2021.04.29.442004 ◽

2021 ◽

Author(s):

Rajan Saha Raju ◽

Abdullah Al Nahid ◽

Preonath Shuvo ◽

Rashedul Islam

Keyword(s):

Genome Sequence ◽

Rna Viruses ◽

Hierarchical Classification ◽

Classification Problem ◽

Virus Genome ◽

Taxonomic Classification ◽

Dna Viruses ◽

Dna And Rna ◽

Full Length Genome

AbstractTaxonomic classification of viruses is a multi-class hierarchical classification problem, as taxonomic ranks (e.g., order, family and genus) of viruses are hierarchically structured and have multiple classes in each rank. Classification of biological sequences which are hierarchically structured with multiple classes is challenging. Here we developed a machine learning architecture, VirusTaxo, using a multi-class hierarchical classification by k-mer enrichment. VirusTaxo classifies DNA and RNA viruses to their taxonomic ranks using genome sequence. To assign taxonomic ranks, VirusTaxo extracts k-mers from genome sequence and creates bag-of-k-mers for each class in a rank. VirusTaxo uses a top-down hierarchical classification approach and accurately assigns the order, family and genus of a virus from the genome sequence. The average accuracies of VirusTaxo for DNA viruses are 99% (order), 98% (family) and 95% (genus) and for RNA viruses 97% (order), 96% (family) and 82% (genus). VirusTaxo can be used to detect taxonomy of novel viruses using full length genome or contig sequences.AvailabilityOnline version of VirusTaxo is available at https://omics-lab.com/virustaxo/.

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Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2110887118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2110887118

Author(s):

Qiang Wang ◽

Lin Zhang ◽

Guo-Wei Zhang ◽

Jian-Hua Mao ◽

Xiao-Dong Xi ◽

...

Keyword(s):

Viral Vectors ◽

Gene Editing ◽

Regulatory Region ◽

Factor Ix ◽

Host Cells ◽

Clotting Factor ◽

Dependent Manner ◽

Coding Region ◽

Therapeutic Benefits

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset–dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

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