scholarly journals Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides

2019 ◽  
Vol 48 (2) ◽  
pp. 802-816 ◽  
Author(s):  
Juergen Scharner ◽  
Wai Kit Ma ◽  
Qian Zhang ◽  
Kuan-Ting Lin ◽  
Frank Rigo ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Genetic Diseases ◽  
Therapeutic Modality ◽  
Target Sequence ◽  
Exon Definition ◽  
Sequence Complementarity ◽  
In Vitro Effects ◽  
Target Activity ◽  
Target Effects

Abstract Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

2019 ◽  
Author(s):  
Yan-ru Cui ◽  
Shao-jie Wang ◽  
Jun Chen ◽  
Jie Li ◽  
Wenzhang Chen ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Therapeutic Agent ◽  
Genetic Diseases ◽  
Ectopic Expression ◽  
Human Cells ◽  
Guide Rna ◽  
Editing Activity ◽  
Periplasmic Domain ◽  
Target Activity

AbstractBackgroundCRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

scholarly journals On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria

2020 ◽  
Vol 48 (11) ◽  
pp. e64-e64 ◽  
Author(s):  
Alicia Calvo-Villamañán ◽  
Jérome Wong Ng ◽  
Rémi Planel ◽  
Hervé Ménager ◽  
Arthur Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
High Throughput Screens ◽  
Target Activity ◽  
Target Effects

Abstract The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

scholarly journals Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

1994 ◽  
Vol 14 (8) ◽  
pp. 5326-5332 ◽  
Author(s):  
J R Perez ◽  
K A Higgins-Sochaski ◽  
J Y Maltese ◽  
R Narayanan
Keyword(s):  
Cell Adhesion ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Antisense Oligonucleotides ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Nf Kappa B ◽  
Growth Factor Beta ◽  
In Vitro Effects

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

scholarly journals Editorial focus: understanding off-target effects as the key to successful RNAi therapy

2019 ◽  
Vol 24 (1) ◽  
Author(s):  
Rafal Bartoszewski ◽  
Aleksander F. Sikorski
Keyword(s):  
Academic Research ◽  
Basic Research ◽  
Drug Candidate ◽  
Related Drug ◽  
Development Pipeline ◽  
Therapy Field ◽  
Target Activity ◽  
Target Effects ◽  
Rnai Therapy

AbstractWith the first RNA interference (RNAi) drug (ONPATTRO (patisiran)) on the market, we witness the RNAi therapy field reaching a critical turning point, when further improvements in drug candidate design and delivery pipelines should enable fast delivery of novel life changing treatments to patients. Nevertheless, ignoring parallel development of RNAi dedicated in vitro pharmacological profiling aiming to identify undesirable off-target activity may slow down or halt progress in the RNAi field. Since academic research is currently fueling the RNAi development pipeline with new therapeutic options, the objective of this article is to briefly summarize the basics of RNAi therapy, as well as to discuss how to translate basic research into better understanding of related drug candidate safety profiles early in the process.

scholarly journals Optimizing sgRNA to Improve CRISPR/Cas9 Knockout Efficiency: Special Focus on Human and Animal Cell

2021 ◽  
Vol 9 ◽  
Author(s):  
Sadegh Shojaei Baghini ◽  
Zhanna R. Gardanova ◽  
Angelina Olegovna Zekiy ◽  
Navid Shomali ◽  
Foad Tosan ◽  
...  
Keyword(s):  
Animal Studies ◽  
High Efficiency ◽  
Animal Cell ◽  
Genetic Diseases ◽  
High Rate ◽  
Special Focus ◽  
Target Sequence ◽  
Human Disorders ◽  
And Function ◽  
Target Effects

During recent years, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies have been noticed as a rapidly evolving tool to deliver a possibility for modifying target sequence expression and function. The CRISPR/Cas9 tool is currently being used to treat a myriad of human disorders, ranging from genetic diseases and infections to cancers. Preliminary reports have shown that CRISPR technology could result in valued consequences for the treatment of Duchenne muscular dystrophy (DMD), cystic fibrosis (CF), β-thalassemia, Huntington’s diseases (HD), etc. Nonetheless, high rates of off-target effects may hinder its application in clinics. Thereby, recent studies have focused on the finding of the novel strategies to ameliorate these off-target effects and thereby lead to a high rate of fidelity and accuracy in human, animals, prokaryotes, and also plants. Meanwhile, there is clear evidence indicating that the design of the specific sgRNA with high efficiency is of paramount importance. Correspondingly, elucidation of the principal parameters that contributed to determining the sgRNA efficiencies is a prerequisite. Herein, we will deliver an overview regarding the therapeutic application of CRISPR technology to treat human disorders. More importantly, we will discuss the potent influential parameters (e.g., sgRNA structure and feature) implicated in affecting the sgRNA efficacy in CRISPR/Cas9 technology, with special concentration on human and animal studies.

scholarly journals We Should Continue the Half-Century History of Applications of Antisense Oligonucleotides in Medicine, Agriculture and Forestry: A Mini-Review

2018 ◽  
Author(s):  
Volodymyr Oberemok ◽  
Kateryna Laikova ◽  
Anna Repetskaya ◽  
Igor Kenyo ◽  
Mikhail Gorlov ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Muscular Atrophy ◽  
Signal To Noise Ratio ◽  
Future Research ◽  
Target Sequence ◽  
Half Century ◽  
Antisense Approach ◽  
History Of ◽  
Post Synthesis

Antisense oligonucleotides (ASO), short single-stranded polymers (based on DNA or RNA chemistries) synthesized in vitro, regulate gene expression by binding in a sequence-specific manner to a RNA target. The functional activity and selectivity in the action of ASOs largely depends on the combination of nitrogenous bases in a target sequence. This simple and natural property of nucleic acids provides an attractive route by which scientists can create different ASO-based techniques. Over the last 50 years, planned and realized applications in the field of antisense technologies have produced astonishing results and posed new challenges for further developments, exemplifying the essence of the post-genomic era. This mini-review critically analyzes some successful cases using the antisense approach in medicine to address severe diseases, such as Duchenne muscular dystrophy and spinal muscular atrophy, and suggests some prospective directions for future research. We also examine in detail the elaboration of insect-specific DNA insecticides and RNA preparations in the areas of agriculture and forestry, a relatively new branch of ASO that allows circumvention of the use of non-selective chemical insecticides. When considering the variety of successful ASO modifications with an efficient signal-to-noise ratio of action, coupled with the affordability of in vitro oligonucleotide synthesis and post-synthesis procedures, we predict that the next half-century will produce a fruitful yield of tools created from effective ASO-based end products.

scholarly journals Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta

1994 ◽  
Vol 14 (8) ◽  
pp. 5326-5332
Author(s):  
J R Perez ◽  
K A Higgins-Sochaski ◽  
J Y Maltese ◽  
R Narayanan
Keyword(s):  
Cell Adhesion ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Antisense Oligonucleotides ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Nf Kappa B ◽  
Growth Factor Beta ◽  
In Vitro Effects

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

scholarly journals A Half-Century History of Applications of Antisense Oligonucleotides in Medicine, Agriculture and Forestry: We Should Continue the Journey

2018 ◽  
Author(s):  
Volodymyr V. Oberemok ◽  
Kateryna V. Laikova ◽  
Anna I. Repetskaya ◽  
Igor M. Kenyo ◽  
Mikhail V. Gorlov ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Muscular Atrophy ◽  
Signal To Noise Ratio ◽  
Future Research ◽  
Target Sequence ◽  
Half Century ◽  
Antisense Approach ◽  
History Of ◽  
Post Synthesis

Antisense oligonucleotides (ASO), short single-stranded polymers based on DNA or RNA chemistries and synthesized in vitro, regulate gene expression by binding in a sequence-specific manner to an RNA target. The functional activity and selectivity in the action of ASOs largely depends on the combination of nitrogenous bases in a target sequence. This simple and natural property of nucleic acids provides an attractive route by which scientists can create different ASO-based techniques. Over the last 50 years, planned and realized applications in the field of antisense and nucleic acid nanotechnologies have produced astonishing results and posed new challenges for further developments, exemplifying the essence of the post-genomic era. Today the majority of ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake. This review critically analyzes some successful cases using the antisense approach in medicine to address severe diseases, such as Duchenne muscular dystrophy and spinal muscular atrophy, and suggests some prospective directions for future research. We also examine in detail the elaboration of unmodified insect-specific DNA insecticides and RNA preparations in the areas of agriculture and forestry, a relatively new branch of ASO that allows circumvention of the use of non-selective chemical insecticides. When considering the variety of successful ASO modifications with an efficient signal-to-noise ratio of action, coupled with the affordability of in vitro oligonucleotide synthesis and post-synthesis procedures, we predict that the next half-century will produce a fruitful yield of tools created from effective ASO-based end products.

Electron Microscopy of Fibroblasts from Myotonic Muscular Dystrophy Patients

1981 ◽  
Vol 39 ◽  
pp. 498-499
Author(s):  
S. E. Miller ◽  
G. B. Hartwig ◽  
R. A. Nielsen ◽  
A. P. Frost ◽  
A. D. Roses
Keyword(s):  
Electron Microscopy ◽  
Muscular Dystrophy ◽  
Genetic Diseases ◽  
Skin Fibroblasts ◽  
Inborn Errors ◽  
Cytoplasmic Inclusions ◽  
Cultured Skin ◽  
Myotonic Muscular Dystrophy ◽  
Glycosaminoglycan Metabolism

Many genetic diseases can be demonstrated in skin cells cultured in vitro from patients with inborn errors of metabolism. Since myotonic muscular dystrophy (MMD) affects many organs other than muscle, it seems likely that this defect also might be expressed in fibroblasts. Detection of an alteration in cultured skin fibroblasts from patients would provide a valuable tool in the study of the disease as it would present a readily accessible and controllable system for examination. Furthermore, fibroblast expression would allow diagnosis of fetal and presumptomatic cases. An unusual staining pattern of MMD cultured skin fibroblasts as seen by light microscopy, namely, an increase in alcianophilia and metachromasia, has been reported; both these techniques suggest an altered glycosaminoglycan metabolism An altered growth pattern has also been described. One reference on cultured skin fibroblasts from a different dystrophy (Duchenne Muscular Dystrophy) reports increased cytoplasmic inclusions seen by electron microscopy. Also, ultrastructural alterations have been reported in muscle and thalamus biopsies from MMD patients, but no electron microscopical data is available on MMD cultured skin fibroblasts.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects
Sign in / Sign up

Export Citation Format

Share Document