One of the distinctive features of the primate genome is the Alu element, a repetitive short interspersed element, over a million highly similar copies of which account for >10% of the genome. A direct consequence of this feature is that primates' transcriptome is highly enriched in long stable dsRNA structures, the preferred target of adenosine deaminases acting on RNAs (ADARs), which are the enzymes catalyzing A‐to‐I RNA editing. Indeed, A‐to‐I editing by ADARs is extremely abundant in primates: over (...) a hundred million editing sites exist in their genomes. However, there are few essential editing sites conserved across mammals that have maintained their editing level despite the radical change in ADAR target landscape. Here, we review and discuss the cost of having an unusual amount of dsRNA and editing in the transcriptome, as well as the opportunities it presents, which might have contributed to the accelerated evolution of the primates. (shrink)

RNA editing is a newly described genetic phenomenon. It encompasses widely different molecular mechanisms and events. According to the specific RNA modification, RNA editing can be broadly classified into six major types. Type II RNA editing occurs in plants and mammals; it consists predominantly in cytidine to uridine conversions resulting from deamination/transamination or transglycosylation, although in plants other mechanisms have not been excluded. Apolipoprotein B mRNA editing is the only well‐documented editing phenomenon in mammals. It (...) is an intranuclear event that occurs posttranscriptionally, coincident with splicing and polyadenylation. Recent observations indicate that the tissue‐ and sequence‐specific process is mediated by an enzyme that has separate domains for editing and sequence recognition. The presence of apolipoprotein B mRNA editing activity in tissues that do not produce the protein suggests that other RNAs may be edited and RNA editing may be a genetic phenomenon of general biological importance to the cell. (shrink)

Genetic variability is considered a key to the evolvability of species. The conversion of an adenosine (A) to inosine (I) in primary RNA transcripts can result in an amino acid change in the encoded protein, a change in secondary structure of the RNA, creation or destruction of a splice consensus site, or otherwise alter RNA fate. Substantial transcriptome and proteome variability is generated by A‐to‐I RNA editing through site‐selective post‐transcriptional recoding of single nucleotides. We posit that this epigenetic source (...) of phenotypic variation is an unrecognized mechanism of adaptive evolution. The genetic variation introduced through editing occurs at low evolutionary cost since predominant production of the wild‐type protein is retained. This property even allows exploration of sequence space that is inaccessible through mutation, leading to increased phenotypic plasticity and provides an evolutionary advantage for acclimatization as well as long‐term adaptation. Furthermore, continuous probing for novel RNA editing sites throughout the transcriptome is an intrinsic property of the editing machinery and represents the molecular basis for increased adaptability. We propose that higher organisms have therefore evolved to systems with increasing RNA editing activity and, as a result, to more complex systems. (shrink)

RNA editing is a major post-transcriptional mechanism that changes specific nucleotides at the RNA level. The most common RNA editing type in humans is adenosine to inosine editing, which is mediated by ADAR enzymes. RNA editing events can not only change amino acids in proteins, but also affect the functions of non-coding RNAs such as miRNAs. Recent studies have characterized thousands of miRNA RNA editing events across different cancer types. Importantly, individual cases of miRNA (...) class='Hi'>editing have been reported to play a role in cancer development. In this review, we summarize the current knowledge of miRNA editing in cancer, and discuss the mechanisms on how miRNA-related editing events modulate the initiation and progression of human cancer. Finally, we discuss the challenges and future directions of studying miRNA editing in cancer. RNA editing is introduced by ADAR or APOBEC enzymes, leading to specific nucleotide changes, respectively. The interplays between RNA editing and miRNAs may play important roles in cancer. This graphical abstract shows four major mechanisms through which miRNAs interact with RNA editing to confer a functional impact on tumor development. (shrink)

Previous studies of Zika virus (ZIKV) pathogenesis have focused primarily on virus‐driven pathology and neurotoxicity, as well as host‐related changes in cell proliferation, autophagy, immunity, and uterine function. It is now hypothesized that ZIKV pathogenesis arises instead as an (unintended) consequence of host innate immunity, specifically, as the side effect of an otherwise well‐functioning machine. The hypothesis presented here suggests a new way of thinking about the role of host immune mechanisms in disease pathogenesis, focusing on dysregulation of post‐transcriptional RNA (...) editing as a candidate driver of a broad range of observed neurodevelopmental defects and neurodegenerative clinical symptoms in both infants and adults linked with ZIKV infections. The authors collect and synthesize existing evidence of ZIKV‐mediated changes in the expression of adenosine deaminases acting on RNA (ADARs), known links between abnormal RNA editing and pathogenesis, as well as ideas for future research directions, including potential treatment strategies. (shrink)

Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X‐ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed (...) considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain‐RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA‐binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs. (shrink)

The most recent work toward compiling a comprehensive database of adenosine‐to‐inosine RNA editing events suggests that the potential for RNA editing is much more pervasive than previously thought; indeed, it is manifest in more than 100 million potential editing events located primarily within Alu repeat elements of the human transcriptome. Pairs of inverted Alu repeats are found in a substantial number of human genes, and when transcribed, they form long double‐stranded RNA structures that serve as optimal substrates (...) for RNA editing enzymes. A small subset of edited Alu elements has been shown to exhibit diverse functional roles in the regulation of alternative splicing, miRNA repression, and cis‐regulation of distant RNA editing sites. The low level of editing for the remaining majority may be non‐functional, yet their persistence in the primate genome provides enhanced genomic flexibility that may be required for adaptive evolution. (shrink)

There is increasing evidence that dynamic changes to chromatin, chromosomes and nuclear architecture are regulated by RNA signalling. Although the precise molecular mechanisms are not well understood, they appear to involve the differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development. A significant fraction of the genome-wide transcription of non-protein coding RNAs may be involved in this process, comprising a previously hidden layer of intermediary genetic information that (...) underpins developmental ontogeny and the differences between species, ecotypes and individuals. It is also evident that RNA editing is a primary means by which hardwired genetic information in animals can be altered by environmental signals, especially in the brain, indicating a dynamic RNA-mediated interplay between the transcriptome, the environment and the epigenome. Moreover, RNA-directed regulatory processes may also transfer epigenetic information not only within cells but also between cells and organ systems, as well as across generations. (shrink)

The evolution of the human brain has resulted in the emergence of higher-order cognitive abilities, such as reasoning, planning and social awareness. Although there has been a concomitant increase in brain size and complexity, and component diversification, we argue that RNA regulation of epigenetic processes, RNA editing, and the controlled mobilization of transposable elements have provided the major substrates for cognitive advance. We also suggest that these expanded capacities and flexibilities have led to the collateral emergence of psychiatric fragilities (...) and conditions. (shrink)

The RNA editing enzyme ADAR1 seemingly has more functions besides RNA editing. Mouse models lacking ADAR1 and sensors of foreign RNA show that RNA editing by ADAR1 plays a crucial role in the innate immune response. Still, RNA editing alone cannot explain all observed phenotypes. Thus, additional roles for ADAR1 must exist. Binding of ADAR1 to RNA is independent of its RNA editing function. Thus, ADAR1 may compete with other RNA-binding proteins. A very recent manuscript (...) elaborates on this and reports competition of ADAR1 with STAUFEN1, thereby modulating RNA-degradation. ADAR1 is also known to recruit proteins such as DROSHA to nascent transcripts. Still, many open questions remain. For instance, the biological role of the Z-DNA binding domains in ADAR1 is not defined. Moreover, the impact of ADAR1 on the RNA-folding landscape is unclear. In sum, moonlighting functions of ADAR1 may be manifold and have a great impact on the transcriptome. Adenosine to inosine RNA editing by ADAR1 is mostly known for its role in the innate immune response to help discriminate self from non-self RNA. Here we discuss the other face of ADAR1 that impacts on cellular processes independent of its editing function. These include Staufen-mediated-decay, mRNA processing, or miRNA maturation. (shrink)

The nomination of candidate genes underlying complex traits is often focused on genetic variations that alter mRNA abundance or result in non‐conservative changes in amino acids. Although inconspicuous in complex trait analysis, genetic variants that affect splicing or RNA editing can also generate proteomic diversity and impact genetic traits. Indeed, it is known that splicing and RNA editing modulate several traits in humans and model organisms. Using high‐throughput RNA sequencing (RNA‐seq) analysis, it is now possible to integrate the (...) genetics of transcript abundance, alternative splicing (AS) and editing with the analysis of complex traits. We recently demonstrated that both AS and mRNA editing are modulated by genetic and environmental factors, and potentially engender phenotypic diversity in a genetically segregating mouse population. Therefore, the analysis of splicing and RNA editing can expand not only the regulatory landscape of transcriptome and proteome complexity, but also the repertoire of candidate genes for complex traits. (shrink)

Most molecular biological concepts derive from physical chemical assumptions about the genetic code that are basically more than 40 years old. Additionally, systems biology, another quantitative approach, investigates the sum of interrelations to obtain a more holistic picture of nucleotide sequence order. Recent empirical data on genetic code compositions and rearrangements by mobile genetic elements and non-coding RNAs, together with results of virus research and their role in evolution, does not really fit into these concepts and compel a re-examination. In (...) this review we try to find an alternate hypothesis. It seems plausible now that if we look at the abundance of regulatory RNAs and persistent viruses in host genomes, we will find more and more evidence that the key players that edit the genetic codes of host genomes are consortia of RNA agents and viruses that drive evolutionary novelty and regulation of cellular processes in all steps of development. This agent-based approach may lead to a qualitative RNA sociology that investigates and identifies relevant behavioural motifs of cooperative RNA consortia. In addition to molecular biological perspectives this may lead to a better understanding of genetic code evolution and dynamics. (shrink)

Recently, constructive neutral evolution has been touted as an important concept for the understanding of the emergence of cellular complexity. It has been invoked to help explain the development and retention of, amongst others, RNA splicing, RNA editing and ribosomal and mitochondrial respiratory chain complexity. The theory originated as a welcome explanation of isolated small scale cellular idiosyncrasies and as a reaction to ‘overselectionism’. Here I contend, that in its extended form, it has major conceptual problems, can not explain (...) observed patterns of complex processes, is too easily dismissive of alternative selectionist models, underestimates the creative force of complexity as such, and – if seen as a major evolutionary mechanism for all organisms – could stifle further thought regarding the evolution of highly complex biological processes. (shrink)

The Streptococcus pyogenes CRISPR‐Cas system has gained widespread application as a genome editing and gene regulation tool as simultaneous cellular delivery of the Cas9 protein and guide RNAs enables recognition of specific DNA sequences. The recent discovery that Cas9 can also bind and cleave RNA in an RNA‐programmable manner indicates the potential utility of this system as a universal nucleic acid‐recognition technology. RNA‐targeted Cas9 (RCas9) could allow identification and manipulation of RNA substrates in live cells, empowering the study of (...) cellular gene expression, and could ultimately spawn patient‐ and disease‐specific diagnostic and therapeutic tools. Here we describe the development of RCas9 and compare it to previous methods for RNA targeting, including engineered RNA‐binding proteins and other types of CRISPR‐Cas systems. We discuss potential uses ranging from live imaging of transcriptional dynamics to patient‐specific therapies and applications in synthetic biology. (shrink)

In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator‐like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)‐associated protein (Cas) system (clustered regularly interspaced short (...) palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR‐Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. (shrink)

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. (shrink)

Graphical AbstractAdenosine Deaminases Acting on RNA (ADARs) enzymes are prominent regulators of neural transcriptome diversity and play a role in the innate immune response. In article number 1800239, Piontkivska et al. outline how neurodevelopmental and neurodegenerative pathogenesis of Zika virus (ZIKV), including congenital Zika and Guillain-Barré syndromes, can be attributed to ADAR editing dysregulation triggered by ZIKV, Explaining Pathogenicity of Congenital Zika and Guillain-Barré Syndromes: Does Dysregulation of RNA Editing Play a Role? DOI: 10.1002/bies.201800239.

The paper argues against the central dogma and its interpretation by C. Kenneth Waters and Alex Rosenberg. I argue that certain phenomena in the regulation of gene expression provide a break with the central dogma, according to which sequence specificity for a gene product must be template derived. My thesis of 'molecular epigenesis' with its three classes of phenomena, sequence 'activation', 'selection', and 'creation', is exemplified by processes such as transcriptional activation, alternative cis- and trans-splicing, and RNA editing. It (...) argues that other molecular resources share the causal role of genes; the sequence specificity for the linear sequence of any gene product is distributed between the coding sequence, cis-acting sequences, trans-acting factors, environmental signals, and the contingent history of the cell (thesis of distributed causal specificity). I conclude that the central dogma has unnecessarily restricted genetic research to the sequencing of protein-coding genes, unilinear pathway analyses, and the focus on exclusive specificity. (shrink)

RNA modifications have recently emerged as an important regulatory layer of gene expression. The most prevalent and reversible modification on messenger RNA (mRNA), N6‐methyladenosine, regulates most steps of RNA metabolism and its dysregulation has been associated with numerous diseases. Other modifications such as 5‐methylcytosine and N1‐methyladenosine have also been detected on mRNA but their abundance is lower and still debated. Adenosine to inosine RNA editing is widespread on coding and non‐coding RNA and can alter mRNA decoding as well as (...) protect against autoimmune diseases. 2′‐O‐methylation of the ribose and pseudouridine are widespread on ribosomal and transfer RNA and contribute to proper RNA folding and stability. While the understanding of the individual role of RNA modifications has now reached an unprecedented stage, still little is known about their interplay in the control of gene expression. In this review we discuss the examples where such interplay has been observed and speculate that with the progress of mapping technologies more of those will rapidly accumulate. (shrink)

Constructive Neutral Evolution is an evolutionary mechanism that can explain much molecular inter-dependence and organismal complexity without assuming positive selection favoring such dependency or complexity, either directly or as a byproduct of adaptation. It differs from but complements other non-selective explanations for complexity, such as genetic drift and the Zero Force Evolutionary Law, by being ratchet-like in character. With CNE, purifying selection maintains dependencies or complexities that were neutrally evolved. Preliminary treatments use it to explain specific genetic and molecular structures (...) or processes, such as retained gene duplications, the spliceosome, and RNA editing. Here we aim to expand the scope of such explanation beyond the molecular level, integrating CNE with Multi-Level Selection theory, and arguing that several popular higher-level selection scenarios are in fact instances of CNE. Suitably contextualized, CNE occurs at any level in the biological hierarchy at which natural selection as normally construed occurs. As examples, we focus on modularity in protein–protein interaction networks or “interactomes,” the origin of eukaryotic cells and the evolution of co-dependence in microbial communities—a variant of the “Black Queen Hypothesis” which we call the “Gray Queen Hypothesis”. (shrink)

Messenger RNA is a flexible tool box that plays a key role in the dynamic regulation of gene expression. RNA modifications variegate the message conveyed by the mRNA. Similar to DNA and histone modifications, mRNA modifications are reversible and play a key role in the regulation of molecular events. Our understanding about the landscape of RNA modifications is still rudimentary in contrast to DNA and histone modifications. The major obstacle has been the lack of sensitive detection methods since they are (...) non-editing events. However, with the advent of next-generation sequencing techniques, RNA modifications are being identified precisely at single nucleotide resolution. In recent years, methylation at the N6 position of adenine has gained the attention of RNA biologists. The m6A modification has a set of writers, erasers, and readers. Here, we provide a summary of interesting facts, conflicting findings, and recent advances in the technical and functional aspects of the m6A epitranscriptome. The recent outburst of studies has brought the key role of mRNA N6-methyladenosine modification in post-transcriptional processing steps into the limelight. This review summarizes the current progress and the future prospective of m6A methylome. We addressed the debatable function of m6A modification and related modifiers in tumorigenesis and tumor progression. (shrink)

Cancer cells exploit mechanisms to evade immune detection triggered by aberrant self‐nucleic acids (NA). PARP7, a key player in this immune evasion strategy, has emerged as a potential target for cancer therapy. PARP7 inhibitors reactivate NA sensing, resulting in type I interferon (IFN) signaling, programmed cell death, anti‐tumor immunity, and tumor regression. Cancer cells with elevated IFN‐stimulated gene (ISG) scores, representing a viral mimicry‐primed state, are particularly sensitive to PARP7 inhibition. This review focuses on the endogenous sources of NA in (...) cancer and the potential to exploit elevated aberrant self‐NA in cancer therapy. We describe strategies to increase cytoplamic NA levels, including targeting epigenetic control, DNA damage response, and mitochondrial function. We also discuss targeting RNA processing pathways, such as splicing and RNA editing, to enhance the immunostimulatory potential of existing NA. Combining PARP7 inhibitors with NA elevating strategies may improve cancer immunotherapy, especially for tumors with high ISG scores. (shrink)

Among pathogenic micro‐organisms that evade the mammalian immune responses, Trypanosoma brucei has developed the most elaborate capacity for antigenic variation. Trypanosomes branched early during eukaryotic evolution. They are characterized by many aberrations, ranging from the unusual compartmentation of metabolic pathways to the heresy of RNA editing. The ubiquitous phenomenon of glycosylphosphatidylinositol‐anchoring of eukaryotic plasma membrane proteins and RNA trans‐splicing (trypanosome genes contain no introns), which adds an identical leader sequence to all trypanosome mRNAs, were first defined during studies of (...) antigenic variation. Genetic transformation of trypanosomes and the high efficiency of gene targeting provide new opportunities to investigate the regulation of antigenic variation. There is every reason to expect trypanosomes to provide further surprises and insights into the evolution of genetic regulatory mechanisms. (shrink)

Mitochondrial genomes represent relict bacterial genomes derived from a progenitor α‐proteobacterium that gave rise to all mitochondria through an ancient endosymbiosis. Evolution has massively reduced these genomes, yet despite relative simplicity their organization and expression has developed considerable novelty throughout eukaryotic evolution. Few organisms have reengineered their mitochondrial genomes as thoroughly as the protist lineage of dinoflagellates. Recent work reveals dinoflagellate mitochondrial genomes as likely the most gene‐impoverished of any free‐living eukaryote, encoding only two to three proteins. The organization and (...) expression of these genomes, however, is far from the simplicity their gene content would suggest. Gene duplication, fragmentation, and scrambling have resulted in an inflated and complex genome organization. Extensive RNA editing then recodes gene transcripts, and trans‐splicing is required to assemble full‐length transcripts for at least one fragmented gene. Even after these processes, messenger RNAs (mRNAs) lack canonical start codons and most transcripts have abandoned stop codons altogether. (shrink)

The multistep pathway of eukaryotic gene expression involves a series of highly regulated events in the nucleus and cytoplasm. In the nucleus, genes are transcribed into pre‐messenger RNAs which undergo a series of nuclear processing steps. Mature mRNAs are then transported to the cytoplasm, where they are translated into protein and degraded at a rate dictated by transcript‐ and cell‐type‐specific cues. Until recently, these individual nuclear and cytoplasmic events were thought to be primarily regulated by different RNA‐ and DNA‐binding proteins (...) that are localized either only in the nucleus or only the cytoplasm. Here, we describe multifunctional proteins that control both nuclear and cytoplasmic steps of gene expression. One such class of multifunctional proteins (e.g., Bicoid and Y‐box proteins) regulates both transcription and translation whereas another class (e.g., Sex‐lethal) regulates both nuclear RNA processing and translation. Other events controlled by multifunctional proteins include assembly of spliceosome components, spliceosome recycling, RNA editing, cytoplasmic mRNA localization, and cytoplasmic RNA stability. The existence of multifunctional proteins may explain the paradoxical involvement of the nucleus in an RNA surveillance pathway (nonsense‐mediated decay) that detects cytoplasmic signals (premature termination codons). We speculate that shuttling multifunctional proteins serve to efficiently link RNA metabolism in the cytoplasmic and nuclear compartments. BioEssays 23:775–787, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

The paper describes the change from molecular genetics to postgenomic biology. It focuses on phenomena in the regulation of gene expression that provide a break with the central dogma, according to which sequence specificity for a gene product must be template derived. In its place we find what is called here ‘constitutive molecular epigenesis’. Its three classes of phenomena, which I call sequence ‘activation’, ‘selection’ and ‘creation’, are exemplified by processes such as transcriptional activation, alternative cis- and trans-splicing, and RNA (...) editing. These phenomena support the following main theses of the paper: 1. Other molecular resources share the causal role of ‘genes’: the ‘causal specificity’ for the linear sequence of any gene product is distributed between the coding sequence, cis-acting sequences, trans-acting factors, environmental signals, and the contingent history of the cell (the cellular code) (thesis of distributed causal specificity). 2. These multiple and overlapping processing and targeting mechanisms amplify the repertoire of RNA and protein products specified through the eukaryotic genome, expanding the possibilities specified by the literal code of DNA (thesis of genetic underdeterminism). 3. These mechanisms of gene expression change the focus of postgenomic research from single molecules and their molecular, biochemical and intrinsic function to their cellular, constituent, component or contextual function due to their recruitment and organization in complex cellular networks. In other words, all agents involved in the regulation of gene expression, including DNA, must interact with other agents to achieve full specificity, which is imposed by regulated recruitment and combinatorial control (theses of regulated recruitment and of system analysis). I conclude from these three main theses that the complexity of higher organisms lies not in its number of genes but in the flexibility, versatility and reactivity of its whole genome. (shrink)

Conventional methods of genetic engineering and more recent genome editing techniques focus on identifying genetic target sequences for manipulation. This is a result of historical concept of the gene which was also the main assumption of the ENCODE project designed to identify all functional elements in the human genome sequence. However, the theoretical core concept changed dramatically. The old concept of genetic sequences which can be assembled and manipulated like molecular bricks has problems in explaining the natural genome-editing (...) competences of viruses and RNA consortia that are able to insert or delete, combine and recombine genetic sequences more precisely than random-like into cellular host organisms according to adaptational needs or even generate sequences de novo. Increasing knowledge about natural genome editing questions the traditional narrative of mutations (error replications) as essential for generating genetic diversity and genetic content arrangements in biological systems. This may have far-reaching consequences for our understanding of artificial genome editing. (shrink)

Large deletions and genomic re‐arrangements are increasingly recognized as common products of double‐strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats ‐ CRISPR associated protein 9 (CRISPR/Cas9) on‐target sites. Together with well‐known off‐target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9‐editing is necessary to ensure validity of observed phenotypes in Cas9‐edited cell‐lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate (...) unwanted effects of Cas9 editing are reviewed; covering guide‐RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off‐targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on‐ and off‐target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms. (shrink)

The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA‐directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site‐specific transcriptional repression or (...) activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. (shrink)

The analysis of genetic and epigenetic mechanisms of the genotype–phenotypic connection has, so far, only been possible in a handful of genetic model systems. Recent technological advances, including next‐generation sequencing methods such as RNA‐seq, ChIP‐seq and RAD‐seq, and genome‐editing approaches including CRISPR‐Cas, now permit to address these fundamental questions of biology also in organisms that have been studied in their natural habitats. We provide an overview of the benefits and drawbacks of these novel techniques and experimental approaches that can (...) now be applied to ecological and evolutionary vertebrate models such as sticklebacks and cichlid fish. We can anticipate that these new methods will increase the understanding of the genetic and epigenetic factors influencing adaptations and phenotypic variation in ecological settings. These new arrows in the methodological quiver of ecologist will drastically increase the understanding of the genetic basis of adaptive traits – leading to a further closing of the genotype–phenotype gap. -/- . (shrink)

Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post‐translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP‐dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP‐A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate (...) H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription‐coupled epigenome editing (or surveillance) function of histone chaperones. (shrink)

With roots in Greek drama, mimesis has recently undergone expansion into an unexpected domain: microbial resistance to viruses. Research revealed that bacteria copy portions of the DNA of attacking viruses and incorporate them into their own DNA. When a virus attacks again, the bacteria generate matching RNA sequences that, together with the Cas9 protein, enable them to recognize the virus and cut its DNA. This process satisfies the requisites for mimesis, thus justifying the name microbiomimesis. It exemplifies nonconscious cognition, a (...) capacity that all lifeforms possess to process and interpret information in contexts that connect it with meaning. In 2012 Jennifer Doudna and Emmanuelle Carpentier coauthored a paper showing that the bacterial defense of RNA/cas9 can be programmed to work as a gene editing tool. So was born CRISPR-Cas9, a technology now routinely used to edit genes by detecting their precise location and modifying them. Never has mimesis been so controversial and laden with implications for the futures of human and nonhuman lifeforms on the planet; what lessons can its traditions offer? (shrink)

Recent experiments have been used to “edit” genomes of various plant, animal and other species, including humans, with unprecedented precision. Furthermore, editing the Cas9 endonuclease gene with a gene encoding the desired guide RNA into an organism, adjacent to an altered gene, could create a “gene drive” that could spread a trait through an entire population of organisms. These experiments represent advances along a spectrum of technological abilities that genetic engineers have been working on since the advent of recombinant (...) DNA techniques. The scientific and bioethics communities have built substantial literatures about the ethical and policy implications of genetic engineering, especially in the age of bioterrorism. However, recent CRISPr/Cas experiments have triggered a rehashing of previous policy discussions, suggesting that the scientific community requires guidance on how to think about social responsibility. We propose a framework to enable analysis of social responsibility, using two example.. (shrink)

The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA‐ and RNA‐targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral (...) nucleic acids in “Point of Care” sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS‐CoV‐2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR‐based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4. (shrink)

How to interpret the “molecular gene” concept is discussed in this paper. I argue that the architecture of biological systems is hierarchical and multi-layered, exhibiting striking similarities to that of modern computers. Multiple layers exist between the genotype and system level property, the phenotype. This architectural complexity gives rise to the intrinsic complexity of the genotype-phenotype relationships. The notion of a gene being for a phenotypic trait or traits lacks adequate consideration of this complexity and has limitations in explaining the (...) genotype-phenotype relationships. I explore ways toward an integrative interpretation of the gene in the context of multi-layered biological systems. A gene, I argue, should be interpreted as a functional unit that is responsible for the trans-generation passage of the capacity to dynamically produce a biochemical activity or biochemical activities. At the molecular level, a gene is a genetic unit, a stretch of DNA sequence, which dictates the behavior and the dynamic production of the encoded cellular component(s). Embedded in a gene’s quadruple DNA code are the regulatory signals, such as those for RNA splicing and/or editing, as well as for transcription factor binding. A regulatory signal can be recognized by the gene expression machinery in one state, but not in another. The confusion caused by RNA splicing, editing, and a gene’s selective tissue distribution pattern is addressed. Instead of a context-dependent definition of the gene, I argue for the view that it is the same gene displaying multiple meanings, subject to differential interpretation by the cellular machinery in different states. In other words, the same gene gives rise to different products and expression levels under different conditions. (shrink)

Volume 8 of this landmark edition follows Peirce from May 1890 through July 1892—a period of turmoil as his career unraveled at the U.S. Coast and Geodetic Survey. The loss of his principal source of income meant the beginning of permanent penury and a lifelong struggle to find gainful employment. His key achievement during these years is his celebrated Monist metaphysical project, which consists of five classic articles on evolutionary cosmology. Also included are reviews and essays from The Nation in (...) which Peirce critiques Paul Carus, William James, Auguste Comte, Cesare Lombroso, and Karl Pearson, and takes part in a famous dispute between Francis E. Abbot and Josiah Royce. Peirce's short philosophical essays, studies in non-Euclidean geometry and number theory, and his only known experiment in prose fiction complete his production during these years. Peirce's 1883-1909 contributions to the Century Dictionary form the content of volume 7 which is forthcoming. (shrink)

Open peer review (OPR) is moving into the mainstream, but it is often poorly understood and surveys of researcher attitudes show important barriers to implementation. As more journals move to implement and experiment with the myriad of innovations covered by this term, there is a clear need for best practice guidelines to guide implementation. This brief article aims to address this knowledge gap, reporting work based on an interactive stakeholder workshop to create best-practice guidelines for editors and journals who wish (...) to transition to OPR. Although the advice is aimed mainly at editors and publishers of scientific journals, since this is the area in which OPR is at its most mature, many of the principles may also be applicable for the implementation of OPR in other areas (e.g., books, conference submissions). (shrink)

Disputatio Vol. V, No. 36_Book Symposium on François Recanati’s Mental Files.