Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model

2019 ◽  
Vol 31 (9) ◽  
pp. 1533 ◽  
Author(s):  
Yu Huang ◽  
Yige Ding ◽  
Yao Liu ◽  
Shiwei Zhou ◽  
Qiang Ding ◽  
...  
Keyword(s):  
Delivery System ◽  
Large Animal ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Sgrna Design ◽  
Large Animals ◽  
Animal Genomes ◽  
Selection Of ◽  
Cas9 Mrna

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL−1 Cas9 mRNA and 25ngμL−1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.

Circulatory Cells as Tumortropic Carrier for Targetability Improvement

2020 ◽  
Vol 17 ◽  
Author(s):  
Dan Zou ◽  
Yajun Weng ◽  
Ping Yang
Keyword(s):  
Drug Delivery ◽  
Drug Delivery System ◽  
Delivery System ◽  
Cell Function ◽  
Incubation Temperature ◽  
Survival Rates ◽  
Controlled Drug Release ◽  
Membrane Receptors ◽  
Circulation Time ◽  
Targeting Efficiency

Background: How to achieve high targeting efficiency for drug delivery system is still one of the most important issues that tumor diagnosis and non-surgical therapies faced. Although nanoparticle-based drug delivery system made an amount of progress in extending circulation time, improving targetability, controlled drug release etc., yet the targeting efficiency remained low, and the development was limited to reduce side effects with overall survival rates unchanged or improved a little. Objective: This paper aims to review current researches on the cell-driven drug delivery systems, and discuss the potential obstacles and directions for cell-based cancer therapies and diagnosis. Methods: More than one hundred references were collected, and this paper focused on red blood cells, monocytes, macrophages, neutrophils, natural killer cells, T lymphocytes, mesenchymal stem cells, cell membrane, artificial cells and extracellular vesicles, then summarized 1) the utilizable properties, 2) balancing cargo-loading amounts and cell function, 3) cascade strategies for targetability improvement. Main findings: circulatory cells and their derivatives were featured by good biocompatibility, long circulation time in blood, unique chemo-migration and penetration ability. On the base of backpack and encapsulation approach, cargo loading amounts and cell function could be balanced through regulating membrane receptors, particle material/size/shape/structure and incubation temperature, etc. The cell-driven drug delivery system met most of the demands that nanoparticle-based delivery system failed to for effective tumortropic delivery. Conclusion: Despite of new challenges, cell-driven drug delivery system generally brought great benefits to and shed a light on for cancer therapy and diagnosis.

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain

2021 ◽  
Vol 332 ◽  
pp. 260-268
Author(s):  
Saed Abbasi ◽  
Satoshi Uchida ◽  
Kazuko Toh ◽  
Theofilus A. Tockary ◽  
Anjaneyulu Dirisala ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Brain ◽  
Guide Rna ◽  
Cas9 Mrna

scholarly journals Review: Tissue Engineering of Small-Diameter Vascular Grafts and Their In Vivo Evaluation in Large Animals and Humans

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 713
Author(s):  
Shu Fang ◽  
Ditte Gry Ellman ◽  
Ditte Caroline Andersen
Keyword(s):  
Animal Models ◽  
Vascular Grafts ◽  
Small Diameter ◽  
Large Animal ◽  
Large Animal Models ◽  
Graft Outcome ◽  
Limited Success ◽  
Large Animals

To date, a wide range of materials, from synthetic to natural or a mixture of these, has been explored, modified, and examined as small-diameter tissue-engineered vascular grafts (SD-TEVGs) for tissue regeneration either in vitro or in vivo. However, very limited success has been achieved due to mechanical failure, thrombogenicity or intimal hyperplasia, and improvements of the SD-TEVG design are thus required. Here, in vivo studies investigating novel and relative long (10 times of the inner diameter) SD-TEVGs in large animal models and humans are identified and discussed, with emphasis on graft outcome based on model- and graft-related conditions. Only a few types of synthetic polymer-based SD-TEVGs have been evaluated in large-animal models and reflect limited success. However, some polymers, such as polycaprolactone (PCL), show favorable biocompatibility and potential to be further modified and improved in the form of hybrid grafts. Natural polymer- and cell-secreted extracellular matrix (ECM)-based SD-TEVGs tested in large animals still fail due to a weak strength or thrombogenicity. Similarly, native ECM-based SD-TEVGs and in-vitro-developed hybrid SD-TEVGs that contain xenogeneic molecules or matrix seem related to a harmful graft outcome. In contrast, allogeneic native ECM-based SD-TEVGs, in-vitro-developed hybrid SD-TEVGs with allogeneic banked human cells or isolated autologous stem cells, and in-body tissue architecture (IBTA)-based SD-TEVGs seem to be promising for the future, since they are suitable in dimension, mechanical strength, biocompatibility, and availability.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Large Animal Models of Vascularized Composite Allotransplantation: A Review of Immune Strategies to Improve Allograft Outcomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Abraham J. Matar ◽  
Rebecca L. Crepeau ◽  
Gerhard S. Mundinger ◽  
Curtis L. Cetrulo ◽  
Radbeh Torabi
Keyword(s):  
Animal Models ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
Tolerance Induction ◽  
Large Animal ◽  
Solid Organ ◽  
Large Animal Models ◽  
Large Animals ◽  
And Function ◽  
Made In

Over the past twenty years, significant technical strides have been made in the area of vascularized composite tissue allotransplantation (VCA). As in solid organ transplantation, the allogeneic immune response remains a significant barrier to long-term VCA survival and function. Strategies to overcome acute and chronic rejection, minimize immunosuppression and prolong VCA survival have important clinical implications. Historically, large animals have provided a valuable model for testing the clinical translatability of immune modulating approaches in transplantation, including tolerance induction, co-stimulation blockade, cellular therapies, and ex vivo perfusion. Recently, significant advancements have been made in these arenas utilizing large animal VCA models. In this comprehensive review, we highlight recent immune strategies undertaken to improve VCA outcomes with a focus on relevant preclinical large animal models.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

SOMES: A REVIEW ON COMPOSITION, FORMULATION METHODS AND EVALUATIONS OF DIFFERENT TYPES OF “SOMES” DRUG DELIVERY SYSTEM

2020 ◽  
pp. 7-18
Author(s):  
KUSUMA PRIYA M. D. ◽  
VINOD KUMAR ◽  
DAMINI V. K. ◽  
ESWAR K. ◽  
KADIRI RAJESH REDDY ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Sustained Release ◽  
Surface Chemistry ◽  
Drug Delivery System ◽  
Delivery System ◽  
Different Types ◽  
Potent Drug ◽  
Evaluation Parameters ◽  
Selection Of

Many drugs are available in the market for several diseases, disorder or even for a condition, but it is difficult to select a suitable carrier to attain maximum bioavailability and potential for a potent drug. Attaining a controlled and sustained release of a drug is purely focused on the selection of a carrier (natural, synthetic and hybrid) like nanosomes. Nanosomes have become a prominent tool in the field of pharmacy. Nanosomes are small uniform structures which deliver the drug to the specific targeted site, which mainly depends upon the presence of ligands, shape, size and surface chemistry. Nanosomes are available in various types which include Niosomes, Liposomes, Electrosomes, Aquasomes, Transfersomes, Phytosomes, Enzymosomes, Ethosomes, Invasome and Sphingosomes. In general, all these nanosomes are quite similar in nature with minute differences in their vesicular characteristics and composition. This review traces various ‘somes’ composition and their role in the formulation, applications, advantages, disadvantages, common formulation procedures and evaluation parameters.

scholarly journals A systematic evaluation on reporting quality of modern studies on pulmonary heart valve implantation in large animals

2020 ◽  
Vol 31 (4) ◽  
pp. 437-445
Author(s):  
Marcelle Uiterwijk ◽  
Annemijn Vis ◽  
Iris de Brouwer ◽  
Debora van Urk ◽  
Jolanda Kluin
Keyword(s):  
Heart Valve ◽  
Heart Valves ◽  
Animal Experiments ◽  
Reporting Quality ◽  
Systematic Evaluation ◽  
Large Animal Model ◽  
Large Animal ◽  
Before And After ◽  
Large Animals

Abstract OBJECTIVES Before new heart valves can be implanted safely in humans, animal experiments have to be performed. These animal experiments have to be clearly designed, analysed and reported to assess the accuracy and importance of the findings. We aimed to provide an overview of the reporting and methodological quality of preclinical heart valve research. METHODS We conducted a systematic literature search on biological and mechanical pulmonary valve implantations in large animals. We used the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines to score the quality of reporting in each article. We compared the scores before and after the introduction of the ARRIVE guidelines (2010). RESULTS We screened 348 articles, of which 31 articles were included. The included articles reported a mean of 54.7% adequately scored ARRIVE items (95% confidence interval 52.2–57.3%). We did not identify a difference in reporting quality (54.7% vs 54.8%) between articles published before and after 2010. We found an unclear (lack of description) risk of selection bias, performance bias and detection bias. CONCLUSIONS The reporting quality of studies that implanted bioprosthetic or mechanical valves in the pulmonary position in the large animal model is not on the desired level. The introduction of the ARRIVE guidelines in 2010 did not improve the reporting quality in this field of research. Hereby, we want to emphasize the importance of clearly describing the methods and transparently reporting the results in animal experiments. This is of great importance for the safe translation of new heart valves to the clinic. Clinical trial registration number PROSPERO (CRD42019147895).

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