Ethical Issues of Using CRISPR Technologies for Research on Military Enhancement

This paper presents an overview of the key ethical questions of performing gene editing research on military service members. The recent technological advance in gene editing capabilities provided by CRISPR/Cas9 and their path towards first-in-human trials has reinvigorated the debate on human enhancement for non-medical purposes. Human performance optimization has long been a priority of military research in order to close the gap between the advancement of warfare and the limitations of human actors. In spite of this focus on temporary performance improvement, biomedical enhancement is an extension of these endeavours and the ethical issues of such research should be considered. In this paper, we explore possible applications of CRISPR to military human gene editing research and how it could be specifically applied towards protection of service members against biological or chemical weapons. We analyse three normative areas including risk–benefit analysis, informed consent, and inequality of access as it relates to CRISPR applications for military research to help inform and provide considerations for military institutional review boards and policymakers.     

基因组编辑技术在精准医学中的应用

基因组编辑技术的飞速发展,尤其是近年来CRISPR/Cas9基因组编辑体系的出现,使得研究人员能高效地在细胞系和动物模型中对基因组进行精确编辑。基于基因组编辑技术的各种实验研究平台被相继开发,包括通过在细胞系中引入疾病相关突变位点建立疾病模型,通过高通量筛选寻找导致肿瘤耐药性的突变基因,通过体内原位靶向致病基因并修改突变进行基因治疗等。这些基因组编辑技术研究平台极大推动了精准医学研究领域的发展。本文对基因组编辑技术在精准医学领域的基础研究、转化应用、目前存在的问题以及未来发展的方向进行了讨论。     

Germline gene editing and the precautionary principle

The precautionary principle aims to influence decision-making in contexts where some activity poses uncertain but potentially grave threats. This perfectly describes the controversy surrounding germline gene editing. This article considers whether the precautionary principle should influence how we weigh the risks and benefits of human germline interventions, focusing especially on the possible threats to the health of future generations. We distinguish between several existing forms of the precautionary principle, assess their plausibility and consider their implications for the ethics of germline modification. We also offer a novel form of the precautionary principle: the sufficientarian precautionary principle. Some plausible versions of the precautionary principle recommend placing somewhat greater weight on avoiding threats to future generations than on achieving short-term benefits. However, no plausible versions of the precautionary principle entail that we should outright reject the use germline gene editing in human reproduction and some, such as the sufficientarian version, might endorse its use.     

The UK’s 100,000 Genomes Project: manifesting policymakers’ expectations

The UK’s 100,000 Genomes Project has the aim of sequencing 100,000 genomes from UK National Health Service (NHS) patients while concomitantly transforming clinical care such that whole genome sequencing becomes routine clinical practice in the UK. Policymakers claim that the project will revolutionize NHS care. We wished to explore the 100,000 Genomes Project, and in particular, the extent to which policymaker claims have helped or hindered the work of those associated with Genomics England – the company established by the Department of Health to deliver the project. We interviewed 20 individuals linked to, or working for Genomics England. Interviewees had double-edged views about the context within which they were working. On the one hand, policymakers’ expectations attached to the venture were considered vacuous “genohype”; on the other hand, they were considered the impetus needed for those trying to advance genomic research into clinical practice. Findings should be considered for future genomes projects.     

Is It Ethical to Use Enhancement Technologies to Make Us Better than Well?

BACKGROUND TO THE DEBATE: A variety of biomedical technologies are being developed that can be used for purposes other than treating disease. Such "enhancement technologies" can be used to improve our appearance and regulate our emotions, with the goal of feeling "better than well." While these technologies can help people adapt to their rapidly changing lifestyles, their use raises important ethical issues.     

Natural Genome Editing from a Biocommunicative Perspective

Natural genome editing from a biocommunicative perspective is the competent agent-driven generation and integration of meaningful nucleotide sequences into pre-existing genomic content arrangements, and the ability to (re-)combine and (re-)regulate them according to context-dependent (i.e. adaptational) purposes of the host organism. Natural genome editing integrates both natural editing of genetic code and epigenetic marking that determines genetic reading patterns. As agents that edit genetic code and epigenetically mark genomic structures, viral and subviral agents have been suggested because they may be evolutionarily older than cellular life. This hypothesis that viruses and viral-like agents edit genetic code is developed according to three well investigated examples that represent key evolutionary inventions in which non-lytic viral swarms act symbiotically in a persistent lifestyle within cellular host genomes: origin of eukaryotic nucleus, adaptive immunity, placental mammals. Additionally an abundance of various RNA elements cooperate in a variety of steps and substeps as regulatory and catalytic units with multiple competencies to act on the genetic code. Most of these RNA agents such as transposons, retroposons and small non-coding RNAs act consortially and are remnants of persistent viral infections that now act as co-opted adaptations in cellular key processes.     

The non-use and influence of UK Energy Sector Indicators

This paper presents the results from a case study on the role in policymaking of UK Energy Sector Indicators (ESIs), introduced by the government in 2003. The findings show that the ESIs constituted a very minor element within the broader evidence-base used by policymakers, and that this indicator set and its objectives were poorly known even to central players in the sector. The findings of this research provide further evidence for the observation that scientific knowledge (including evaluations, assessments and indicators) seldom play an instrumental role in policymaking, and are more likely to produce indirect, conceptual and political impacts. The analysis provides a number of tentative conclusions concerning such potential indirect impacts that accrue mainly through processes of dialogue and argumentation both during the preparation of the indicators and after their publication as part of the annual reporting by the UK energy department. The ESIs have played various conceptual and political roles, yet the concrete outcomes in terms of policy change remain to be explored. The conclusions highlight the limitations of rationalist notions of direct, instrumental use in the efforts to understand the role of indicators in policymaking. The paper concludes by three tentative propositions concerning the explanations to the absence of instrumental role of the ESIs, which could be usefully explored in future research: the characteristics of the energy sector; the characteristics of the UK policy culture; and the exceptionality of the ESIs in the general evidence-base of UK energy sector.     

CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool

With the emergence of CRISPR technology, targeted editing of a wide variety of genomes is no longer an abstract hypothetical, but occurs regularly. As application areas of CRISPR are exceeding beyond research and biomedical therapies, new and existing ethical concerns abound throughout the global community about the appropriate scope of the systems' use. Here we review fundamental ethical issues including the following: 1) the extent to which CRISPR use should be permitted; 2) access to CRISPR applications; 3) whether a regulatory framework(s) for clinical research involving human subjects might accommodate all types of human genome editing, including editing of the germline; and 4) whether international regulations governing inappropriate CRISPR utilization should be crafted and publicized. We conclude that moral decision making should evolve as the science of genomic engineering advances and hold that it would be reasonable for national and supranational legislatures to consider evidence-based regulation of certain CRISPR applications for the betterment of human health and progress.     

Genomic imprinting and mammalian reproduction

Among animals, genomic imprinting is a uniquely mammalian phenomenon in which certain genes are monoallelically expressed according to their parent of origin. This silencing of certain alleles often involves differential methylation at regulatory regions associated with imprinted genes and must be recapitulated at every generation with the erasure and reapplication of these epigenetic marks in the germline. Imprinted genes encode regulatory proteins that play key roles in fetal growth and development, but they also exert wider effects on mammalian reproduction. Genetic knockout experiments have shown that certain paternally expressed imprinted genes regulate post-natal behavior in offspring as well as reproductive behaviors in males and females. These deficits involve changes in hypothalamic function affecting multiple areas and different neurochemical pathways. Paternally expressed genes are highly expressed in the hypothalamus which regulates growth, metabolism and reproduction and so are well placed to influence all aspects of reproduction from adults to the resultant offspring. Coadaptation between offspring and mother appears to have played an important role in the evolution of some paternally expressed genes, but the influence of these genes on male reproductive behavior also suggests that they have evolved to regulate their own transmission to successive generations via the male germline.     

Biocommunication and natural genome editing

The biocommunicative approach investigates communication processes within and among cells, tissues, organs and organisms as sign-mediated interactions, and nucleotide sequences as code, i.e. language-like text, which follows in parallel three kinds of rules: combinatorial (syntactic), context-sensitive (pragmatic), and content-specific (semantic). Natural genome editing from a biocommunicative perspective is competent agent-driven generation and integration of meaningful nucleotide sequences into pre-existing genomic content arrangements and the ability to (re-)combine and (re-)regulate them according to context-dependent (i.e. adaptational) purposes of the host organism.     

Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize “off-target” sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.     

Impact of methodological choices on assessments of the reliability of fossil primate phylogenetic hypotheses

It has been argued in several recent studies that conventional craniodental characters cannot be assumed to be reliable for the purposes of reconstructing primate phylogenetic relationships and that as a consequence little confidence can be invested in published fossil primate phylogenies. Here, we evaluate this claim by revisiting the analyses reported in one of these studies [Collard and Wood, 2000]. Specifically, we investigate whether the use of alternative methodological procedures would have altered their findings. We focus on three key issues: (1) size correction, (2) outgroup composition and (3) non-phylogenetic correlation among characters. Our analyses suggest that the results of Collard and Wood [2000] were not affected by the size correction method they used or by the outgroup they employed. Our analyses also suggest that their results were not affected by their decision to ignore developmental, functional and other non-phylogenetic correlations among the characters in their data sets. Accordingly, our study supports the assertion that conventional craniodental characters cannot be assumed to be reliable for reconstructing primate phylogenetic relationships. This in turn suggests that many published fossil primate phylogenies may be unreliable.     

Mammalian conserved ADAR targets comprise only a small fragment of the human editosome

Background

ADAR proteins are among the most extensively studied RNA binding proteins. They bind to their target and deaminate specific adenosines to inosines. ADAR activity is essential, and the editing of a subset of their targets is critical for viability. Recently, a huge number of novel ADAR targets were detected by analyzing next generation sequencing data. Most of these novel editing sites are located in lineage-specific genomic repeats, probably a result of overactivity of editing enzymes, thus masking the functional sites. In this study we aim to identify the set of mammalian conserved ADAR targets.

Results

We used RNA sequencing data from human, mouse, rat, cow, opossum, and platypus to define the conserved mammalian set of ADAR targets. We found that the conserved mammalian editing sites are surprisingly small in number and have unique characteristics that distinguish them from non-conserved ones. The sites that constitute the set have a distinct genomic distribution, tend to be located in genes encoding neurotransmitter receptors or other synapse related proteins, and have higher editing and expression levels. We also found a high consistency of editing levels of this set within mice strains and between human and mouse. Tight regulation of editing in these sites across strains and species implies their functional importance.

Conclusions

Despite the discovery of numerous editing targets, only a small number of them are conserved within mammalian evolution. These sites are extremely highly conserved and exhibit unique features, such as tight regulation, and probably play a pivotal role in mammalian biology.     

Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.     

Campylobacter bacteriophages and bacteriophage therapy

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.