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RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

Transformative research is a broad and loosely connected family of research disciplines and approaches, with the explicit normative ambition to fundamentally question the status quo, change the dominant structures, and support just sustainability transitions by working collaboratively with society. When engaging in such science-practice collaborations for transformative change in society, researchers experience ethical dilemmas. Amongst others, they must decide, what is worthwhile to be researched, whose reality is privileged, and whose knowledge is included. Yet, current institutionalised ethical standards, which largely follow the tradition of medical ethics, are insufficient to guide transformative researchers in navigating such dilemmas. In addressing this vacuum, the research community has started to develop peer guidance on what constitutes morally good behaviour. These formal and informal guidelines offer a repertoire to explain and justify positions and decisions. However, they are only helpful when they have become a part of researchers’ practical knowledge ‘in situ’. By focusing on situated research practices, the article addresses the need to develop an attitude of leaning into the uncertainty around what morally good behaviour constitutes. It also highlights the significance of combining this attitude with a critical reflexive practice both individually and collaboratively for answering questions around ‘how to’ as well as ‘what is the right thing to do’. Using a collaborative autoethnographic approach, the authors of this paper share their own ethical dilemmas in doing transformative research, discuss those, and relate them to a practical heuristic encompassing axiological, ontological, and epistemological considerations. The aim is to support building practical wisdom for the broader research community about how to navigate ethical questions arising in transformative research practice.

Transformative research is a broad and loosely connected family of research disciplines and approaches, with the explicit normative ambition to fundamentally question the status quo, change the dominant structures, and support just sustainability transitions by working collaboratively with society. When engaging in such science-practice collaborations for transformative change in society, researchers experience ethical dilemmas. Amongst others, they must decide, what is worthwhile to be researched, whose reality is privileged, and whose knowledge is included. Yet, current institutionalised ethical standards, which largely follow the tradition of medical ethics, are insufficient to guide transformative researchers in navigating such dilemmas. In addressing this vacuum, the research community has started to develop peer guidance on what constitutes morally good behaviour. These formal and informal guidelines offer a repertoire to explain and justify positions and decisions. However, they are only helpful when they have become a part of researchers’ practical knowledge ‘in situ’. By focusing on situated research practices, the article addresses the need to develop an attitude of leaning into the uncertainty around what morally good behaviour constitutes. It also highlights the significance of combining this attitude with a critical reflexive practice both individually and collaboratively for answering questions around ‘how to’ as well as ‘what is the right thing to do’. Using a collaborative autoethnographic approach, the authors of this paper share their own ethical dilemmas in doing transformative research, discuss those, and relate them to a practical heuristic encompassing axiological, ontological, and epistemological considerations. The aim is to support building practical wisdom for the broader research community about how to navigate ethical questions arising in transformative research practice.

Transformative research is a broad and loosely connected family of research disciplines and approaches, with the explicit normative ambition to fundamentally question the status quo, change the dominant structures, and support just sustainability transitions by working collaboratively with society. When engaging in such science-practice collaborations for transformative change in society, researchers experience ethical dilemmas. Amongst others, they must decide, what is worthwhile to be researched, whose reality is privileged, and whose knowledge is included. Yet, current institutionalised ethical standards, which largely follow the tradition of medical ethics, are insufficient to guide transformative researchers in navigating such dilemmas. In addressing this vacuum, the research community has started to develop peer guidance on what constitutes morally good behaviour. These formal and informal guidelines offer a repertoire to explain and justify positions and decisions. However, they are only helpful when they have become a part of researchers’ practical knowledge ‘in situ’. By focusing on situated research practices, the article addresses the need to develop an attitude of leaning into the uncertainty around what morally good behaviour constitutes. It also highlights the significance of combining this attitude with a critical reflexive practice both individually and collaboratively for answering questions around ‘how to’ as well as ‘what is the right thing to do’. Using a collaborative autoethnographic approach, the authors of this paper share their own ethical dilemmas in doing transformative research, discuss those, and relate them to a practical heuristic encompassing axiological, ontological, and epistemological considerations. The aim is to support building practical wisdom for the broader research community about how to navigate ethical questions arising in transformative research practice.

This paper in using a novel application of the Delphi panel method, explores and consolidates the future visions of a low carbon and climate resilient future in Ireland through community-based visioning processes that emerged through co-created, deliberative approaches at local level. It embraces a visioning process that applies a transdisciplinary approach, which aims to match different visions and stakeholder needs, combining bottom-up and top-down perspectives. Community visions were articulated by means of a series of deliberative futures workshops facilitated by the transdisciplinary project research team. Through iterations of the Delphi feedback loop, the notion of deep and shallow agreement is explored to examine the spectrum of consensus to dissent around the co-developed future visions and pathways of climate action, articulated by the Irish community case study. This approach enables a critical exploration of the conventional science-policy-practice nexus through the use of innovative creative communication and engagement methods. Findings suggest that more deliberative forums which bring together a diverse range of stakeholders are needed to develop a shared vision for the future; the importance of language use and the creation of a shared vocabulary of climate action; and the power of civic imaginaries in helping to create useful future narratives.

This paper in using a novel application of the Delphi panel method, explores and consolidates the future visions of a low carbon and climate resilient future in Ireland through community-based visioning processes that emerged through co-created, deliberative approaches at local level. It embraces a visioning process that applies a transdisciplinary approach, which aims to match different visions and stakeholder needs, combining bottom-up and top-down perspectives. Community visions were articulated by means of a series of deliberative futures workshops facilitated by the transdisciplinary project research team. Through iterations of the Delphi feedback loop, the notion of deep and shallow agreement is explored to examine the spectrum of consensus to dissent around the co-developed future visions and pathways of climate action, articulated by the Irish community case study. This approach enables a critical exploration of the conventional science-policy-practice nexus through the use of innovative creative communication and engagement methods. Findings suggest that more deliberative forums which bring together a diverse range of stakeholders are needed to develop a shared vision for the future; the importance of language use and the creation of a shared vocabulary of climate action; and the power of civic imaginaries in helping to create useful future narratives.

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to "proofreading" capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

Transition discourses are gaining prominence in efforts to imagine a future that adequately addresses the urgent need to establish low carbon and climate resilient pathways. Within these discourses the 'public' is seen as central to the creation and implementation of appropriate interventions. The role of public engagement in societal transformation while essential, is also complex and often poorly understood. The purpose of this paper is to enhance our understanding regarding public engagement and to address the often superficial and shallow policy discourse on this topic. The paper offers a review of evolving literature to map emergent public engagement in processes of transition and change. We adopt a pragmatic approach towards literature retrieval and analysis which enables a cross-disciplinary and cross-sectoral review. We use a scoping review process and the three spheres of transformation framework (designated as the practical, political and personal spheres) to explore trends within this complex research field. The review draws from literature from the last two decades in the Irish context and looks at emergence and evolving spaces of public engagement within various systems of change including energy, food, coastal management and flood adaptation, among others.

Transition discourses are gaining prominence in efforts to imagine a future that adequately addresses the urgent need to establish low carbon and climate resilient pathways. Within these discourses the 'public' is seen as central to the creation and implementation of appropriate interventions. The role of public engagement in societal transformation while essential, is also complex and often poorly understood. The purpose of this paper is to enhance our understanding regarding public engagement and to address the often superficial and shallow policy discourse on this topic. The paper offers a review of evolving literature to map emergent public engagement in processes of transition and change. We adopt a pragmatic approach towards literature retrieval and analysis which enables a cross-disciplinary and cross-sectoral review. We use a scoping review process and the three spheres of transformation framework (designated as the practical, political and personal spheres) to explore trends within this complex research field. The review draws from literature from the last two decades in the Irish context and looks at emergence and evolving spaces of public engagement within various systems of change including energy, food, coastal management and flood adaptation, among others.

Powerful new technologies for perturbing genetic elements have recently expanded the study of genetic interactions in model systems ranging from yeast to human cell lines. However, technical artifacts can confound signal across genetic screens and limit the immense potential of parallel screening approaches. To address this problem, we devised a novel PCA-based method for correcting genome-wide screening data, bolstering the sensitivity and specificity of detection for genetic interactions. Applying this strategy to a set of 436 whole genome CRISPR screens, we report more than 1.5 million pairs of correlated "co-functional" genes that provide finer-scale information about cell compartments, biological pathways, and protein complexes than traditional gene sets. Lastly, we employed a gene community detection approach to implicate core genes for cancer growth and compress signal from functionally related genes in the same community into a single score. This work establishes new algorithms for probing cancer cell networks and motivates the acquisition of further CRISPR screen data across diverse genotypes and cell types to further resolve complex cellular processes.

Powerful new technologies for perturbing genetic elements have recently expanded the study of genetic interactions in model systems ranging from yeast to human cell lines. However, technical artifacts can confound signal across genetic screens and limit the immense potential of parallel screening approaches. To address this problem, we devised a novel PCA-based method for correcting genome-wide screening data, bolstering the sensitivity and specificity of detection for genetic interactions. Applying this strategy to a set of 436 whole genome CRISPR screens, we report more than 1.5 million pairs of correlated "co-functional" genes that provide finer-scale information about cell compartments, biological pathways, and protein complexes than traditional gene sets. Lastly, we employed a gene community detection approach to implicate core genes for cancer growth and compress signal from functionally related genes in the same community into a single score. This work establishes new algorithms for probing cancer cell networks and motivates the acquisition of further CRISPR screen data across diverse genotypes and cell types to further resolve complex cellular processes.

The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.

The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA-target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9-DNA interaction and broaden the toolkit for investigating off-target binding behavior.

Several recent papers have reported strong signals of selection on European polygenic height scores. These analyses used height effect estimates from the GIANT consortium and replication studies. Here, we describe a new analysis based on the the UK Biobank (UKB), a large, independent dataset. We find that the signals of selection using UKB effect estimates are strongly attenuated or absent. We also provide evidence that previous analyses were confounded by population stratification. Therefore, the conclusion of strong polygenic adaptation now lacks support. Moreover, these discrepancies highlight (1) that methods for correcting for population stratification in GWAS may not always be sufficient for polygenic trait analyses, and (2) that claims of differences in polygenic scores between populations should be treated with caution until these issues are better understood.Editorial noteThis article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The accumulation of amyloid beta (A beta) peptide (Amyloid cascade hypothesis), an APP protein cleavage product, is a leading hypothesis in the etiology of Alzheimer's disease (AD). In order to identify additional AD risk genes, we performed targeted sequencing and rare variant burden association study for nine candidate genes involved in the amyloid metabolism in 1886 AD cases and 1700 controls. We identified a significant variant burden association for the gene encoding caspase-8, CASP8 (p = 8.6x10(-5)). For two CASP8 variants, p.K148R and p.I298V, the association remained significant in a combined sample of 10,820 cases and 8,881 controls. For both variants we performed bioinformatics structural, expression and enzymatic activity studies and obtained evidence for loss of function effects. In addition to their role in amyloid processing, caspase-8 and its downstream effector caspase-3 are involved in synaptic plasticity, learning, memory and control of microglia pro-inflammatory activation and associated neurotoxicity, indicating additional mechanisms that might contribute to AD. As caspase inhibition has been proposed as a mechanism for AD treatment, our finding that AD-associated CASP8 variants reduce caspase function calls for caution and is an impetus for further studies on the role of caspases in AD and other neurodegenerative diseases.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

The accumulation of amyloid beta (A beta) peptide (Amyloid cascade hypothesis), an APP protein cleavage product, is a leading hypothesis in the etiology of Alzheimer's disease (AD). In order to identify additional AD risk genes, we performed targeted sequencing and rare variant burden association study for nine candidate genes involved in the amyloid metabolism in 1886 AD cases and 1700 controls. We identified a significant variant burden association for the gene encoding caspase-8, CASP8 (p = 8.6x10(-5)). For two CASP8 variants, p.K148R and p.I298V, the association remained significant in a combined sample of 10,820 cases and 8,881 controls. For both variants we performed bioinformatics structural, expression and enzymatic activity studies and obtained evidence for loss of function effects. In addition to their role in amyloid processing, caspase-8 and its downstream effector caspase-3 are involved in synaptic plasticity, learning, memory and control of microglia pro-inflammatory activation and associated neurotoxicity, indicating additional mechanisms that might contribute to AD. As caspase inhibition has been proposed as a mechanism for AD treatment, our finding that AD-associated CASP8 variants reduce caspase function calls for caution and is an impetus for further studies on the role of caspases in AD and other neurodegenerative diseases.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

Detection of recent natural selection is a challenging problem in population genetics. Here we introduce the singleton density score (SDS), a method to infer very recent changes in allele frequencies from contemporary genome sequences. Applied to data from the UK10K Project, SDS reflects allele frequency changes in the ancestors of modern Britons during the past ~2000 to 3000 years. We see strong signals of selection at lactase and the major histocompatibility complex, and in favor of blond hair and blue eyes. For polygenic adaptation, we find that recent selection for increased height has driven allele frequency shifts across most of the genome. Moreover, we identify shifts associated with other complex traits, suggesting that polygenic adaptation has played a pervasive role in shaping genotypic and phenotypic variation in modern humans.

Focal malformations of cortical development, including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are important causes of intractable childhood epilepsy. Using targeted and exome sequencing on DNA from resected brain samples and nonbrain samples from 53 patients with FCD or HME, we identified pathogenic germline and mosaic mutations in multiple PI3K/AKT pathway genes in 9 patients, and a likely pathogenic variant in 1 additional patient. Our data confirm the association of DEPDC5 with sporadic FCD but also implicate this gene for the first time in HME. Our findings suggest that modulation of the mammalian target of rapamycin pathway may hold promise for malformation-associated epilepsy.

Focal malformations of cortical development, including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are important causes of intractable childhood epilepsy. Using targeted and exome sequencing on DNA from resected brain samples and nonbrain samples from 53 patients with FCD or HME, we identified pathogenic germline and mosaic mutations in multiple PI3K/AKT pathway genes in 9 patients, and a likely pathogenic variant in 1 additional patient. Our data confirm the association of DEPDC5 with sporadic FCD but also implicate this gene for the first time in HME. Our findings suggest that modulation of the mammalian target of rapamycin pathway may hold promise for malformation-associated epilepsy.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive mid-hindbrain malformation. JS is part of a group of disorders called ciliopathies based on their overlapping phenotypes and common underlying pathophysiology linked to primary cilium dysfunction. Biallelic mutations in one of 28 genes, all encoding proteins localizing to the primary cilium or basal body, can cause JS. Despite this large number of genes, the genetic cause can currently be determined in about 62% of individuals with JS. To identify novel JS genes, we performed whole exome sequencing on 35 individuals with JS and found biallelic rare deleterious variants (RDVs) in KIAA0586, encoding a centrosomal protein required for ciliogenesis, in one individual. Targeted next-generation sequencing in a large JS cohort identified biallelic RDVs in eight additional families for an estimated prevalence of 2.5% (9/366 JS families). All affected individuals displayed JS phenotypes toward the mild end of the spectrum.