Developments In Gene Editing Research Articles

Extrinsic and intrinsic regulation of blood feeding in mosquitoes.

Mosquitoes obtain large amounts of blood from hosts in a short period of time. To efficiently obtain high-quality blood without being noticed by the host, mosquitoes sense external factors such as the taste of the host blood and the surrounding environment, and integrate these signals with their own internal information to determine whether to initiate blood feeding and how long to continue feeding. With the development of gene editing and behavior monitoring techniques, the factors that control blood feeding are being identified. Elucidating the factors that contribute to blood feeding is expected to provide new ideas for artificially controlling blood feeding, which has often been overlooked behind host attraction mechanisms. Furthermore, understanding salivary components, mechanisms controlling satiety in feeding, and differences between sugar feeding and blood feeding would help us understand how some mosquitoes have adopted and developed blood feeding over the course of evolution.

Digital data storage on DNA tape using CRISPR base editors

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA’s enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called “DNA Mutational Overwriting Storage” (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school’s logo and the title of this study on the DNA tapes.

Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

Perspectives on biotechnology: Public and corporate narratives in the GM archives

Societal Impact StatementThe contentious debate over genetically modified (GM) crops in Britain has entered a new era following Brexit and the development of gene editing. At the same time, the events of the 1980s and 1990s are now entering historical archives, including the GM Archive at the Science Museum in London. This article explores how fears of unnaturalness and arguments from historical continuity informed the British GM controversy. It also analyses the limitations of these modern archives. The concerns surrounding the naturalness and continuity of biotechnology have recently been resurrected following suggestions that gene editing will be employed in plant breeding.Summary This research examines the introduction of genetically modified (GM) crops to Britain, including the backlash from environmentalist groups and the public that led to a de facto government moratorium on their commercialisation in 1998. Harnessing archival materials from the Science Museum's GM Archive, this paper shows that GM was viewed as an alien or unnatural technology in Britain, while campaigns from Monsanto and scientific supporters of GM attempted to show how recombinant DNA technology was simply the latest step in a long history of plant breeding. By moving outside the archive, it becomes clear that the creation of this narrative from continuity was a standard industry strategy. Appeals to the history of plant breeding was a strategy adopted by Monsanto well before the British GM controversy, while twentieth‐century food scares had already undermined public trust in government and industry. Public concern over the naturalness of biotechnology remains. Meanwhile, contemporary advocates of gene editing have begun to make similar arguments to those deployed in the 1990s, highlighting the similarity of gene editing to natural variation and selection.

Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

CRISPR systems: what’s new, where next?

The genetic signature of natural CRISPR-Cas systems were first noted in a 1989 publication and were characterized in detail from 2002 to 2007, culminating in the first report of a prokaryotic adaptive immune system. Since then, CRISPR-Cas enzymes have been adapted into molecular biology tools that have transformed genetic engineering across domains of life. In this feature article, we describe origins, uses and futures of CRISPR-Cas enzymes in genetic engineering: we highlight advances made in the past 10 years. Central to these advances is appreciation of interplay between CRISPR engineering and DNA repair. We highlight how this relationship has been manipulated to create further advances in the development of gene editing.

TALEN-mediated generation of Nkx3.1 knockout rat model.

Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans. Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology. Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors. We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.

The new landscape of retinal gene therapy.

Novel therapeutics for inherited retinal dystrophies (IRDs) have rapidly evolved since groundbreaking clinical trials for LCA due to RPE65 mutations led to the first FDA-approved in vivo gene therapy. Since then, advancements in viral vectors have led to more efficient AAV transduction and developed other viral vectors for gene augmentation therapy of large gene targets. Furthermore, significant developments in gene editing and RNA modulation technologies have introduced novel capabilities for treatment of autosomal dominant diseases, intronic mutations, and/or large genes otherwise unable to be treated with current viral vectors. We highlight strategies currently being evaluated in gene therapy clinical trials and promising preclinical developments for IRDs.

New developments in gene editing for Duchenne muscular dystrophy

A somatic gene editing therapy for the treatment of Duchenne muscular dystrophy (DMD) improves skeletal and cardiac muscle function and reduces cardiac arrhythmogenic vulnerability in a pig model of DMD and an in vitro model of human DMD.

Blurring the boundaries between cereal crops and model plants.

The cereal crops rice (Oryza sativa), maize (Zea mays ssp.mays) and wheat (Triticum aestivum) provide half of the food eaten by humankind. However, understanding their biology has proved challenging due to their large size, long lifecycle and large genomes. The model plant Arabidopsis thaliana avoids these practical problems and has provided fundamental understanding of plant biology, however not all of this knowledge is directly transferrable to cereals. Recent developments in gene editing, speed breeding and genome assembly techniques mean that the challenges associated with working with the major cereal crops can be overcome. Resources such as mutant collections and genome sequences are now available for these crops, making them attractive experimental systems with which to make discoveries that are directly applicable to increasing crop production.

Human germline gene editing. Recommendations of the European Society of Human Genetics and the European Society of Human Reproduction and Embryology.*

Technological developments in gene editing raise high expectations for clinical applications, first of all somatic gene editing but in theory also germline gene editing. The latter is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and European Society of Human Genetics (ESHG) together developed a Background Document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Board and Executive Committee of the Societies in April/May 2017. Taking account of ethical arguments, it is argued that both basic and preclinical research regarding human germline gene editing can be justified on conditions. Furthermore, while clinical germline gene editing would be totally premature, it might become a responsible intervention in the future, but only after adequate preclinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as preimplantation genetic diagnosis and somatic editing if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

Technological developments in gene editing raise high expectations for clinical applications, including editing of the germline. The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. This document provides the background to the Recommendations. Germline gene editing is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique could help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? This Background document summarizes the scientific developments and expectations regarding germline gene editing, legal regulations at the European level, and ethics for three different settings (basic research, preclinical research and clinical applications). In ethical terms, we argue that the deontological objections (e.g., gene editing goes against nature) do not seem convincing while consequentialist objections (e.g., safety for the children thus conceived and following generations) require research, not all of which is allowed in the current legal situation in European countries. Development of this Background document and Recommendations reflects the responsibility to help society understand and debate the full range of possible implications of the new technologies, and to contribute to regulations that are adapted to the dynamics of the field while taking account of ethical considerations and societal concerns.

Human germline gene editing: Recommendations of ESHG and ESHRE

Technological developments in gene editing raise high expectations for clinical applications, first of all for somatic gene editing but in theory also for germline gene editing (GLGE). GLGE is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if GLGE would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Executive Committee and the Board of the respective Societies in May 2017. Taking account of ethical arguments, we argue that both basic and pre-clinical research regarding GLGE can be justified, with conditions. Furthermore, while clinical GLGE would be totally premature, it might become a responsible intervention in the future, but only after adequate pre-clinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as PGD and somatic editing, if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome.

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications.

Responsible innovation in human germline gene editing. Background document to the recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, including editing of the germline. The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. This document provides the background to the Recommendations. Germline gene editing is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique could help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? This Background document summarizes the scientific developments and expectations regarding germline gene editing, legal regulations at the European level, and ethics for three different settings (basic research, pre-clinical research and clinical applications). In ethical terms, we argue that the deontological objections (e.g. gene editing goes against nature) do not seem convincing while consequentialist objections (e.g. safety for the children thus conceived and following generations) require research, not all of which is allowed in the current legal situation in European countries. Development of this Background document and Recommendations reflects the responsibility to help society understand and debate the full range of possible implications of the new technologies, and to contribute to regulations that are adapted to the dynamics of the field while taking account of ethical considerations and societal concerns.

2017 Year In Review: From Immunotherapy to Developments in Gene Editing, Experts in the Field Weigh in on the Year's Most Important Advances

2017 Year In Review: From Immunotherapy to Developments in Gene Editing, Experts in the Field Weigh in on the Year's Most Important Advances

Human germline gene editing. Recommendations of ESHG and ESHRE.

Technological developments in gene editing raise high expectations for clinical applications, first of all for somatic gene editing but in theory also for germline gene editing (GLGE). GLGE is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if GLGE would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and the European Society of Human Genetics (ESHG) together developed a Background document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Executive Committee and the Board of the respective Societies in May 2017. Taking account of ethical arguments, we argue that both basic and pre-clinical research regarding human GLGE can be justified, with conditions. Furthermore, while clinical GLGE would be totally premature, it might become a responsible intervention in the future, but only after adequate pre-clinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as PGD and somatic editing, if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

Induced pluripotent stem cell technology: a decade of progress.

Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modelling, drug discovery and cell therapy development. Novel pathological mechanisms have been elucidated, new drugs originating from iPSC screens are in the pipeline and the first clinical trial using human iPSC-derived products has been initiated. In particular, the combination of human iPSC technology with recent developments in gene editing and 3D organoids makes iPSC-based platforms even more powerful in each area of their application, including precision medicine. In this Review, we discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, and consider the remaining challenges and the emerging opportunities in the field.

The transgenic animal platform for biopharmaceutical production.

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.