scholarly journals Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing

2019 ◽  
Vol 11 (11) ◽  
pp. 10472-10480 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
Katie Sanders ◽  
Jordan J. Green
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Dna And Rna

scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

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.

scholarly journals A paradoxical synergism between Resveratrol and copper (II) with respect to degradation of DNA and RNA

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1145 ◽  
Author(s):  
Siddharth Subramaniam ◽  
Iqbal Vohra ◽  
Aishwarya Iyer ◽  
Naveen K Nair ◽  
Indraneel Mittra
Keyword(s):  
Synergistic Effect ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Oxygen Species ◽  
Molar Ratios ◽  
Dna Cleaving ◽  
Dna And Rna ◽  
Fixed Concentration ◽  
Anti Cancer ◽  
Low Levels

Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical synergistic effect between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical synergistic effect is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

scholarly journals Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery

2018 ◽  
Vol 10 (38) ◽  
pp. 31915-31927 ◽  
Author(s):  
Yuyuan Wang ◽  
Ben Ma ◽  
Amr A. Abdeen ◽  
Guojun Chen ◽  
Ruosen Xie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Messenger Rna ◽  
Redox Responsive

scholarly journals Advances in biotechnology: Genomics and genome editing

2017 ◽  
Vol 1 (1) ◽  
pp. 2-9
Author(s):  
Kevan M.A. Gartland ◽  
Munis Dundar ◽  
Tommaso Beccari ◽  
Mariapia Viola Magni ◽  
Jill S. Gartland
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Disease Transmission ◽  
Gene Editing ◽  
Genome Project ◽  
Sequence Information ◽  
Regulatory Processes ◽  
Dna And Rna ◽  
Mitochondrial Replacement Therapy ◽  
The Uk

Abstract Genomics, the study of genes, their functions and related techniques has become a crucial science for developing understanding of life processes and how they evolve. Since the advent of the human genome project, huge strides have been made in developing understanding of DNA and RNA sequence information and how it can be put to good use in the biotechnology sector. Newly derived sequencing and bioinformatics tools have added to the torrent of new insights gained, so that ‘sequence once and query often’ type DNA apps are now becoming reality. Genome editing, using tools such as CRISPR/Cas9 nuclease or Cpf1 nuclease, provide rapid methods for inserting, deleting or modifying DNA sequences in highly precise ways, in virtually any animal, plant or microbial system. Recent international discussions have considered human germline gene editing, amongst other aspects of this technology. Whether or not gene edited plants will be considered as genetically modified remains an important question. This will determine the regulatory processes adopted by different groups of nations and applicability to feeding the world’s ever growing population. Questions surrounding the intellectual property rights associated with gene editing must also be resolved. Mitochondrial replacement therapy leading to ‘3-Parent Babies’ has been successfully carried out in Mexico, by an international team, to correct mother to child mitochondrial disease transmission. The UK has become the first country to legally allow ‘cautious use’ of mitochondrial donation in treatment. Genomics and genome editing will continue to advance what can be achieved technically, whilst society determines whether or not what can be done should be applied.

scholarly journals The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

2021 ◽  
Author(s):  
Aleksia Barka ◽  
Kiara N. Berríos ◽  
Peter Bailer ◽  
Emily K. Schutsky ◽  
Tong Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Strong Dependence ◽  
Cytosine Deamination ◽  
Base Editing ◽  
Dna And Rna ◽  
Dna Backbone ◽  
Mechanistic Basis ◽  
Cellular Dna ◽  
Editing Enzyme

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

scholarly journals Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA

Cell Reports ◽  
2022 ◽  
Vol 38 (1) ◽  
pp. 110196
Author(s):  
Xianming Zhang ◽  
Hua Jin ◽  
Xiaojia Huang ◽  
Birendra Chaurasiya ◽  
Daoyin Dong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Nanoparticle Delivery

Abstract 528: Towards Therapeutic Genome Editing in Vascular Endothelium by Novel Nanoparticle Delivery of Crispr Plasmid Dna

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xianming Zhang ◽  
You-yang Zhao
Keyword(s):  
Protein Expression ◽  
Genome Editing ◽  
Vascular Endothelium ◽  
Plasmid Dna ◽  
Adult Life ◽  
Therapeutic Delivery ◽  
Crispr System ◽  
Highly Efficient ◽  
Adult Mice

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice. Methods: We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter (EC-specific) and gRNA by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

scholarly journals Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl

2019 ◽  
Vol 63 (6-7) ◽  
pp. 281-286 ◽  
Author(s):  
Toshinori Hayashi ◽  
Mie Nakajima ◽  
Mitsuki Kyakuno ◽  
Kanako Doi ◽  
Ikumi Manabe ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Genome Editing ◽  
Plasmid Dna ◽  
Experimental System ◽  
Gene Manipulation ◽  
Experimental Animals ◽  
The Core ◽  
Research Fields ◽  
Injection Protocol ◽  
Pleurodeles Waltl

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

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