scholarly journals Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Eman A. Ageely ◽  
Ramadevi Chilamkurthy ◽  
Sunit Jana ◽  
Leonora Abdullahu ◽  
Daniel O’Reilly ◽  
...  
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Model Organisms ◽  
Enzyme Properties ◽  
Chemical Compatibility ◽  
Modified Nucleotides ◽  
Chemical Modifications ◽  
Pseudoknot Structure ◽  
Therapeutic Development

AbstractCRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

scholarly journals Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Puneet Sharma ◽  
Jie Wu ◽  
Benedikt S. Nilges ◽  
Sebastian A. Leidel
Keyword(s):  
Human Cells ◽  
Ribosome Profiling ◽  
Model Organisms ◽  
Treatment Conditions ◽  
Genome Wide ◽  
Optimized Protocol ◽  
Gene Level ◽  
Translation Inhibitor ◽  
Species Specific ◽  
Conformational Restrictions

AbstractRibosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

scholarly journals CRISPR gene-editing creates wave of exotic model organisms

Nature ◽  
2019 ◽  
Vol 568 (7753) ◽  
pp. 441-442 ◽  
Author(s):  
Sara Reardon
Keyword(s):  
Gene Editing ◽  
Model Organisms

scholarly journals Gene editing

2018 ◽  
Vol 64 (1) ◽  
pp. 9-12
Author(s):  
Piotr Węgleński
Keyword(s):  
Genetic Engineering ◽  
Gene Editing ◽  
Social Problems ◽  
Metabolic Diseases ◽  
Genetic Diseases ◽  
Model Organisms ◽  
Wild Type ◽  
Gene Engineering ◽  
Working Out ◽  
Asilomar Conference

Development of the gene engineering techniques has raised worries that they will be used for construction of organism endangering humansand environment. In 1975 at the Asilomar conference, geneticists from many countries decided that genetic engineering brings more benefitsthan threats. In last years a new CRISPR-Cas technique emerged . It allows to make the precise changes in genomes, e.g. to inactivate particulargenes or to replace mutated genes by their wild-type alleles. Inactivation in mice of genes corresponding to those whose mutations causethe genetic diseases in man allows to get model organisms for studying the etiology of given disease and for working out the methods of itscuring. This technique can be applied for repairing genes whose mutations result in metabolic diseases and cancer. Some voices were raisedthat the technique can be potentially used for the “improvement” of man, what would create many ethical and social problems. Geneticists,ethicists and lawyers gathered in 2015 at the Washington conference, discussed these problems and proposed rules for their solving.

scholarly journals The Future of Gene-Guided Neuroscience Research in Non-Traditional Model Organisms

2019 ◽  
Vol 93 (2-3) ◽  
pp. 108-121 ◽  
Author(s):  
Scott Juntti
Keyword(s):  
Gene Editing ◽  
High Throughput Sequencing ◽  
Model Organism ◽  
Model Organisms ◽  
Traditional Model ◽  
Model Species ◽  
Neuroscience Research ◽  
Experimental Approaches ◽  
Natural Variations ◽  
And Behavior

Natural variations across animals in form, function, and behavior have long been sources of inspiration to scientists. Despite this, experimentalists focusing on the neural bases of behavior have increasingly focused on a select few model species. This consolidation is motivated primarily by the availability of resources and technologies for manipulation in these species. Recent years have witnessed a proliferation of experimental approaches that were developed primarily in traditional model species, but that may in principle be readily applied to any species. High-throughput sequencing, CRISPR gene editing, transgenesis, and other technologies have enabled new insights through their deployment in non-traditional model species. The availability of such approaches changes the calculation of which species to study, particularly when a trait of interest is most readily observed in a non-traditional model organism. If these technologies are widely adopted in many new species, it promises to revolutionize the field of neuroethology.

scholarly journals Ethical and regulatory aspects of genome editing

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2553-2560 ◽  
Author(s):  
Donald B. Kohn ◽  
Matthew H. Porteus ◽  
Andrew M. Scharenberg
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Clinical Implementation ◽  
Hematopoietic Stem ◽  
Regulatory Aspects ◽  
Regulatory Frameworks ◽  
Therapeutic Development ◽  
Important Field ◽  
Developing Area ◽  
Effective Use

Abstract Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic.

scholarly journals An optimized base editor with efficient C-to-T base editing in zebrafish

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yu Zhao ◽  
Dantong Shang ◽  
Ruhong Ying ◽  
Hanhua Cheng ◽  
Rongjia Zhou
Keyword(s):  
Gene Editing ◽  
Cytidine Deaminase ◽  
Model Organism ◽  
Model Organisms ◽  
Base Substitution ◽  
Base Editing ◽  
Codon Preference ◽  
Promising Tool ◽  
Human Genes ◽  
Target Sites

Abstract Background Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Results Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Conclusions Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

scholarly journals SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

2015 ◽  
Vol 89 ◽  
pp. 379-386 ◽  
Author(s):  
Kimberly Cramer-Morales ◽  
Collin D. Heer ◽  
Kranti A. Mapuskar ◽  
Frederick E. Domann
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Targeted Gene Editing

scholarly journals CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ashley J. Clark ◽  
Niamh Mullooly ◽  
Dewi Safitri ◽  
Matthew Harris ◽  
Tessa de Vries ◽  
...  
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Calcitonin Receptor ◽  
Physiological Relevance ◽  
Starting Point ◽  
Calcitonin Gene ◽  
Ramp Function ◽  
Physiological Pathways ◽  
Cardiovascular Cells ◽  
Primary Human Cells

AbstractAgonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

scholarly journals Evolutionary analyses guide selection of model systems to investigate proto-oncogene function in ALK and LTK

2019 ◽  
Author(s):  
Zheng Wang ◽  
Alex Dornburg ◽  
Junrui Wang ◽  
Elizabeth S. Mo ◽  
Francesc Lopez-Giraldez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Anaplastic Lymphoma Kinase ◽  
Model Systems ◽  
Model Organisms ◽  
Tyrosine Kinase Receptors ◽  
Evolutionary Analysis ◽  
Anaplastic Lymphoma ◽  
Evolutionary Innovation ◽  
Therapeutic Development ◽  
Selection Of

AbstractModel systems to investigate oncogene-driven cancer have played an essential role in the development of therapies for cancer. However, not all systems are appropriate for all therapeutic targets. Knowing where and when proto-oncogenes and their interactors originated in evolutionary history is key to understanding which organisms can serve as models. Here we investigate two tyrosine kinase receptors that underlie tumorigenesis in cancer: anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK). In Drosophila melanogaster, Caenorhabitis elegans, and Homo sapiens, the discovery of putative ligands Jeb, Hen-1, and AUG has the potential to accelerate the development of novel therapeutics. However, homology of these ligands and receptors is unclear. We performed an exhaustive search for their homologs spanning the metazoan tree of life. Jeb and Hen-1 were restricted to species that diverged prior to the origin of all vertebrates. No non-vertebrate species had ligands orthologous to AUG. Instead, an ancestral receptor tyrosine kinase and AUG gene were present in early vertebrates and are still solitary in lamprey; we demonstrate that the early embryonic expression of AUG in lamprey parallels its expression in model mammal systems. The presence of ALK and LTK in jawed vertebrates is an evolutionary innovation, as is a previously unrecognized functional convergence within ALK and LTK occurring between actinopterygians and sarcopterygians. Our results provide the phylogenetic context necessary for the selection of model organisms that will provide informative investigations of the biology of these critically important tyrosine kinase receptors, enabling successful therapeutic development.SignificanceThe anaplastic lymphoma kinase ALK can be oncogenically altered to become a driver of several malignancies, including non-small-cell lung cancer and anaplastic large-cell lymphomas. The development of therapeutics targeting this gene depend on the discovery of its interacting partner ligands in relevant model organisms. ALK is found across most major animal groups including mammals, fishes, and invertebrates. Correspondly, several candidate ligands for ALK and its duplicate LTK have been advanced by research in model species. However their homology to the human ligands and therefore their potential to guide therapeutic development is unknown. Our comparative evolutionary analysis revealed which model organisms had functional receptor-ligand pairings that are informative regarding the role of these genes in human tumorigenesis.

Sign in / Sign up

Export Citation Format

Share Document