Use Of Genome Editing Research Articles

Screening for gene doping transgenes in horses via the use of massively parallel sequencing.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Thinking the unthinkable: how did human germline genome editing become ethically acceptable?

Two major reports in the UK and USA have recently sanctioned as ethically acceptable genome editing of future generations for the treatment of serious rare inherited conditions. This marks an important turning point in the application of recombinant DNA techniques to humans. The central question this paper addresses is how did it became possible for human genetic engineering (HGE) of future generations to move from an illegitimate idea associated with eugenics in the 1980s to a concrete proposal sanctioned by scientists and bioethicists in 2020? The paper uses the concept of a regime of normativity to understand the co-evolution and mutual shaping of technology, imaginaries, norms and governance processes in debates about HGE in the USA and UK. It will be argued that interlinked discursive, institutional, political and technological changes have made proposals for the use of genome editing in the genetic engineering of future generations both “thinkable” and legitimate.

The role of externalities and uncertainty in policy design: evidence from the regulation of genome editing

Externalities and uncertainty play an important role in the design of regulatory policies. Regulatory tools must be selected while taking into consideration the side-effects that regulated products or services have on other individuals and on the environment. This study investigates the externalities and uncertainty that arise from the use of genome editing (with specific reference to CRISPR technique) and how they relate to regulatory policy design choices. Building on evidence from genome editing regulation and on the NATO (Nodality, Authority, Treasure and Organization) policy tools framework, this study argues that a mix of regulatory tools is required to tackle externalities of genome editing applications and to cope with sources of uncertainty about their beneficial, neutral and harmful side-effects. The study provides some recommendations to policy-makers about reducing uncertainty, diversifying regulatory tools over time, and communicating to the public about features of genetically edited products.

Fetal Hemoglobin (HbF) Silencer BCL11A Acts through a Novel DNA-Motif in the Gamma-Globin Promoters, Simplifying the Model for Hemoglobin Switching

Fetal hemoglobin (HbF) level is under strong genetic control. Common genetic variation is associated with modest elevation in HbF levels in adults. Genome-wide association studies led to identification of the regulatory factor BCL11A, which is a major contributor to HbF silencing. Despite clear evidence for its involvement, how BCL11A acts to repress gamma-globin gene expression has remained unknown. Conflicting data exist in the literature with regard to whether BCL11A binds DNA directly and, if so, the sequence(s) it may recognize. Prior experiments from our laboratory suggested that BCL11A acts at a distance from the gamma-globin gene promoters, but failed to define with confidence sites of occupancy within the beta globin locus. To explore mechanisms by which BCL11A functions, we first devised a cellular assay for rescue of BCL11A-deficient erythroid cells with BCL11A cDNA constructs. This approach established that of the two longer BCL11A isoforms (L and XL), only XL (“eXtraLong”) is competent to repress globin expression in cells. XL differs from L by the presence of an alternatively spliced terminal exon encoding three closely spaced zinc-fingers (ZF4-6), implicating this domain in an essential function of the protein. Given that ZFs often mediate sequence-specific DNA-binding, we employed a Protein Binding Microarray platform (PBM) that allows for high-throughput determination of transcription factor binding specificities at high-throughput on an oligonucleotide matrix. We determined that recombinant ZF4-6, as well as BCL11A-XL, binds sequence-specifically to a novel consensus motif with a central core, TGACCA. Two biophysical methods confirmed recognition of this motif by recombinant ZF4-6 with an affinity of ~ 25nM. The motif is present in promoters of all fetal or embryonic-expressed globin genes in human and mouse, and in two copies in each human gamma-globin gene promoter (-119 to -113 and -92 to -86). The motif is altered by single base substitutions in rare HPFH alleles (-117 and -114) and by a microdeletion HPFH (-114 to -102). HPFH-associated single base substitutions greatly impair ZF4-6 binding, therefore strongly implicating a direct role for BCL11A at the gamma-globin gene promoters. However, numerous attempts to localize endogenous BCL11A protein in chromatin of erythroid cells by conventional chromatin immunoprecipitation sequencing (ChIP-seq) proved unsuccessful. To determine if the gamma-globin gene promoters are indeed bound by BCL11A in vivo in erythroid cells, we adapted a newly described method for detection of protein occupancy in chromatin (CUT&RUN) at higher resolution and without crosslinking that is commonly used in ChIP-seq. With this method, we detected BCL11A occupancy in the gamma-globin gene promoters in adult erythroid cells, including CD34 cells, and moreover observed that the distal one of the repeated motifs is the predominant binding site in vivo . Taken together, these experiments provide persuasive evidence that BCL11A acts locally at the gamma-globin gene promoters to repress transcription and that classical HPFH alleles in the (-117 to -114) region lead to increased HbF due to failure of BCL11A binding. These findings are consistent with a revised model of HbF silencing in which BCL11A acts through direct gamma-globin promoter binding, but do not exclude additional architectural or long-distance roles of BCL11A in HbF silencing. The critical role of the BCL11A DNA-binding motif in mediating HbF repression further supports the rationale for use of genome editing to target this region as therapy of the hemoglobinopathies. DisclosuresOrkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy.

Genome Editing in Livestock, Complicity, and the Technological Fix Objection

Genome editing in livestock could potentially be used in ways that help resolve some of the most urgent and serious global problems pertaining to livestock, including animal suffering, pollution, antimicrobial resistance, and the spread of infectious disease. But despite this potential, some may object to pursuing it, not because genome editing is wrong in and of itself, but because it is the wrong kind of solution to the problems it addresses: it is merely a ‘technological fix’ to a complex societal problem. Yet though this objection might have wide intuitive appeal, it is often not clear what, exactly, the moral problem is supposed to be. The aim of this paper is to formulate and shed some light on the ‘technological fix objection’ to genome editing in livestock. I suggest that three concerns may underlie it, make implicit assumptions underlying the concerns explicit, and cast some doubt on several of these assumptions, at least as they apply to the use of genome editing to produce pigs resistant to the Porcine Reproductive and Respiratory Syndrome and hornless dairy cattle. I then suggest that the third, and most important, concern could be framed as a concern about complicity in factory farming. I suggest ways to evaluate this concern, and to reduce or offset any complicity in factory farming. Thinking of genome editing’s contribution to factory farming in terms of complicity, may, I suggest, tie it more explicitly and strongly to the wider obligations that come with pursuing it, including the cessation of factory farming, thereby addressing the concern that technological fixes focus only on a narrow problem.

Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies

Therapeutic advances for neurological disorders are challenging due to limited accessibility of the human central nervous system and incomplete understanding of disease mechanisms. Many neurological diseases lack precision treatments, leading to significant disease burden and poor outcome for affected patients. Induced pluripotent stem cell (iPSC) technology provides human neuronal cells that facilitate disease modeling and development of therapies. The use of genome editing, in particular CRISPR-Cas9 technology, has extended the potential of iPSCs, generating new models for a number of disorders, including Alzheimers and Parkinson Disease. Editing of iPSCs, in particular with CRISPR-Cas9, allows generation of isogenic pairs, which differ only in the disease-causing mutation and share the same genetic background, for assessment of phenotypic differences and downstream effects. Moreover, genome-wide CRISPR screens allow high-throughput interrogation for genetic modifiers in neuronal phenotypes, leading to discovery of novel pathways, and identification of new therapeutic targets. CRISPR-Cas9 has now evolved beyond altering gene expression. Indeed, fusion of a defective Cas9 (dCas9) nuclease with transcriptional repressors or activation domains allows down-regulation or activation of gene expression (CRISPR interference, CRISPRi; CRISPR activation, CRISPRa). These new tools will improve disease modeling and facilitate CRISPR and cell-based therapies, as seen for epilepsy and Duchenne muscular dystrophy. Genome engineering holds huge promise for the future understanding and treatment of neurological disorders, but there are numerous barriers to overcome. The synergy of iPSC-based model systems and gene editing will play a vital role in the route to precision medicine and the clinical translation of genome editing-based therapies.

Making sense of heritable human genome editing: Scientific and ethical considerations.

Genome editing, particularly the use of CRISPR-Cas9-based methodologies, is revolutionizing biology through its impacts on research and the translation of these into applications in biomedicine. Somatic genome editing aimed at treating individuals with disease raises some significant ethical issues, but proposed heritable interventions, through the use of genome editing in gametes or embryos, raise a number of distinct social, ethical and political issues. This review will consider some proposed uses of heritable human genome editing (HHGE) and several of the objections to these that have been raised. Making sense of such proposed uses requires viewing HHGE as an assisted reproductive technology (ART) that, like preimplantation genetic testing (PGT) and mitochondrial replacement techniques (MRT), aims to prevent disease transmission during sexual reproduction, rather than acting as a therapy for an existing individual. Applications beyond the paradigm of disease prevention raise even more difficult scientific and ethical questions. Here, I will discuss various themes that are prominent in discussions of the science and ethics of HHGE, including impacts on human dignity and society, the language of HHGE used for public dialogue and the governance of HHGE.

120 The future of genome editing in food animal species

Abstract Genome editing refers to the use of targeted nucleases to introduce double-stranded breaks in DNA at a specific sequence in the genome. The cell can then repair this break using either the error-prone, non-homologous end joining (NHEJ) pathway which often results in a small insertion or deletion. This can inactivate or “knock-out” a gene if the repair introduces a missense or nonsense frameshift mutation in the coding region of the protein encoded by that gene. Alternatively, the break can be repaired via the homology-directed repair (HDR) pathway which relies on a nucleic acid template to guide the repair. Depending upon the template, the directed repair can be as simple as a single nucleotide change, or as complicated as an entire transgene integration. Editing offers an opportunity for geneticists to precisely introduce useful genetic alterations into animal breeding programs. Some examples include a knock-out of the CD163 cellular receptor of porcine reproductive and respiratory syndrome virus (PRRSV) which renders pigs resistant to infection by that virus, and the replacement of the horned allele in dairy cattle with the Celtic (PC) POLLED allele resulting in genetic hornlessness. For editing to be useful, it will need to seamlessly integrate with genetic improvement program design. This will ultimately require introducing edits into multiple elite lines to avoid genetic bottlenecks. This requirement is at odds with the process-based trigger and event-based regulatory approach that has been proposed for gene edited food animals by the US Food and Drug Administration (FDA). The 2017 draft FDA regulatory guidance 187 states that “intentional” genomic alterations introduced by genome editing will be regulated as new animal drugs, irrespective of product novelty or risk, necessitating lengthy multigenerational safety evaluations. The high costs associated with new animal drug approval will dramatically curtail the use of genome editing in food animal species.

Genome Edited Crops Touch the Market: A View on the Global Development and Regulatory Environment.

Products of genome editing as the most promising “New Plant Breeding Technology” (NPBT) have made the transition from the lab to the market in a short time. Globally, research activities employing genome editing are constantly expanding and more and more plants with market-oriented traits are being developed, and companies have already released the first genome edited crops to the market. Few countries, most of which are located in the Americas, have adapted legislations to these technologies or released guidelines supporting the use of genome editing. Other countries are debating the path to come either because there is no clarity on the legal classification or due consensus is hampered by a renewed GMO debate. In recent years (2017−2020), eight countries have introduced guidelines clarifying the legal status of genome edited products and many of those are actively committed to international harmonization of their policies. In this publication we give an overview on the current and potentially future international regulatory environment and an update on plants derived by genome editing with market-oriented traits.

Issues in the Clinical Use of Genome Editing in Human Embryos From a Medical Perspective

Issues in the Clinical Use of Genome Editing in Human Embryos From a Medical Perspective

Human Germ Line and Heritable Genome Editing: The Global Policy Landscape.

Discussions and debates about the governance of human germline and heritable genome editing should be informed by a clear and accurate understanding of the global policy landscape. This policy survey of 106 countries yields significant new data. A large majority of countries (96 out of 106) surveyed have policy documents-legislation, regulations, guidelines, codes, and international treaties-relevant to the use of genome editing to modify early-stage human embryos, gametes, or their precursor cells. Most of these 96 countries do not have policies that specifically address the use of genetically modified in vitro embryos in laboratory research (germline genome editing); of those that do, 23 prohibit this research and 11 explicitly permit it. Seventy-five of the 96 countries prohibit the use of genetically modified in vitro embryos to initiate a pregnancy (heritable genome editing). Five of these 75 countries provide exceptions to their prohibitions. No country explicitly permits heritable human genome editing. These data contrast markedly with previously reported findings.

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

BackgroundGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques.ResultsIn the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated.ConclusionsThe optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

Introgression Breeding in Barley: Perspectives and Case Studies.

Changing production scenarios resulting from unstable climatic conditions are challenging crop improvement efforts. A deeper and more practical understanding of plant genetic resources is necessary if these assets are to be used effectively in developing improved varieties. In general, current varieties and potential varieties have a narrow genetic base, making them prone to suffer the consequences of new and different abiotic and biotic stresses that can reduce crop yield and quality. The deployment of genomic technologies and sophisticated statistical analysis procedures has generated a dramatic change in the way we characterize and access genetic diversity in crop plants, including barley. Various mapping strategies can be used to identify the genetic variants that lead to target phenotypes and these variants can be assigned coordinates in reference genomes. In this way, new genes and/or new alleles at known loci present in wild ancestors, germplasm accessions, land races, and un-adapted introductions can be located and targeted for introgression. In principle, the introgression process can now be streamlined and linkage drag reduced. In this review, we present an overview of (1) past and current efforts to identify diversity that can be tapped to improve barley yield and quality, and (2) case studies of our efforts to introgress resistance to stripe and stem rust from un-adapted germplasm. We conclude with a description of a modified Nested Association Mapping (NAM) population strategy that we are implementing for the development of multi-use naked barley for organic systems and share perspectives on the use of genome editing in introgression breeding.

Genetic Protection Modifications: Moving Beyond the Binary Distinction Between Therapy and Enhancement for Human Genome Editing.

The current debate and policy surrounding the use of genome editing in humans typically relies on a binary distinction between therapy and human enhancement. Here, we argue that this dichotomy fails to take into account perhaps the most significant potential uses of CRISPR-Cas9 genome editing in humans. We argue that genetic treatment of sporadic Alzheimer's disease, breast and ovarian cancer predisposing BRCA1/2 mutations, and the introduction of human immunodeficiency virus resistance in humans should be considered within a new category of genetic protection treatments. We suggest that if this category is not introduced, life-altering research might be unnecessarily limited by current or future policy. Otherwise ad hoc decisions might be made, which introduce a risk of unforeseen moral costs, and might overlook or fail to address some important opportunities.

Recent advances in developing disease resistance in plants.

Approaches to manipulating disease resistance in plants is expanding exponentially due to advances in our understanding of plant defense mechanisms and new tools for manipulating the plant genome. The application of effective strategies is only limited now by adoption of rapid classical genetic techniques and the acceptance of genetically engineered traits for some problems. The use of genome editing and cis-genetics, where possible, may facilitate applications that otherwise require considerable time or genetic engineering, depending on settling legal definitions of the products. Nonetheless, the variety of approaches to developing disease resistance has never been greater.

Genome Editing Animals and the Promise of Control in a (Post-) Anthropocentric World

Gene editing tools are ‘revolutionizing’ microbiological research. Much of the public debate focuses on the possibility of human germ line applications. The use of genome editing to alter non-human animals, however, will have more immediate impacts on our daily lives. Genome edited animals are used for basic biological and biomedical research and could soon play a role in the livestock industry and ecosystem management. Genome editing thus provides an occasion to rethink societal narratives about the relationships between humans and other animals. Even though the technique can be easily incorporated as an example into a conventional storyline about the development of the modern life sciences as striving for control over nature, it can also help to highlight the anthropocentric biases expressed in these narratives and demonstrate the continuities between humans and other animals.

Application of genome editing in farm animals: cattle.

Milk and meat from cattle and buffaloes contribute 45% of the global animal protein supply, followed by chickens (31%), and pigs (20%). In 2016, the global cattle population of 1.0 billion head produced 6.5 billion tons of cows' milk, and 66 million tons of beef. In the past century, cattle breeding programs have greatly increased the yield per animal with a resultant decrease in the emissions intensity per unit of milk or beef, but this has not been true in all regions. Genome editing research in cattle to date has focused on disease resistance (e.g. tuberculosis), production (e.g. myostatin knockout; production of all-male offspring), elimination of allergens (e.g. beta-lactoglobulin knockout) and welfare (e.g. polled or hornlessness) traits. Modeling has revealed how the use of genome editing to introduce beneficial alleles into cattle breeds could maintain or even accelerate the rate of genetic gain accomplished by conventional breeding programs, and is a superior approach to the lengthy process of introgressing those same alleles from distant breeds. Genome editing could be used to precisely introduce useful alleles (e.g. heat tolerance, disease resistance) and haplotypes into native locally-adapted cattle breeds, thereby helping to improve their productivity. As with earlier genetic engineering approaches, whether breeders will be able to employ genome editing in cattle genetic improvement programs will very much depend upon global decisions around the regulatory framework and governance of genome editing for food animals.

CRISPR: a promising tool for lipid physiology and therapeutics.

Purpose of reviewThe purpose is to review recent progress in applying the CRISPR/Cas9 system to lipid metabolism and therapeutics.Recent findingsThe CRISPR/Cas9 system has been used to generate knockout animals for lipid genes in multiple species. Somatic genome editing with CRISPR/Cas9 can efficiently disrupt genes in adult animals, including a new strategy for generating atherosclerosis. Refinements to the CRISPR/Cas9 system including epigenetic modulators and base editors offer new avenues to manipulate gene expression. The recent report of germline genome editing in humans highlights the promise as well as perils of this technology.SummaryCRISPR/Cas9 is a transformative technology that will help advance on our understanding of lipid metabolism and physiology. Somatic genome editing is a particularly promising approach for editing genes in tissues of live organisms, and represents a new means of addressing unmet therapeutic challenges in humans. Educational outreach, public debate, and consideration of ethics and safety must guide the use of genome editing in humans.

Engineering of high-precision base editors for site-specific single nucleotide replacement

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable.

CRISPRing the Elephant in the Room

The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host & Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium.