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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.

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

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.

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).

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.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

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 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.

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.

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).

Disparities for women and minorities in science, technology, engineering, and math (STEM) careers have continued even amidst mounting evidence for the superior performance of diverse workforces. In response, we launched the Diversity and Science Lecture series, a cross-institutional platform where junior life scientists present their research and comment on diversity, equity, and inclusion in STEM. We characterize speaker representation from 79 profiles and investigate topic noteworthiness via quantitative content analysis of talk transcripts. Nearly every speaker discussed interpersonal support, and three-fifths of speakers commented on race or ethnicity. Other topics, such as sexual and gender minority identity, were less frequently addressed but highly salient to the speakers who mentioned them. We found that significantly co-occurring topics reflected not only conceptual similarity, such as terms for racial identities, but also intersectional significance, such as identifying as a Latina/Hispanic woman or Asian immigrant, and interactions between concerns and identities, including the heightened value of friendship to the LGBTQ community, which we reproduce using transcripts from an independent seminar series. Our approach to scholar profiles and talk transcripts serves as an example for transmuting hundreds of hours of scholarly discourse into rich datasets that can power computational audits of speaker diversity and illuminate speakers' personal and professional priorities. [ABSTRACT FROM AUTHOR]

Functional consequences of genetic variants are best studied by manipulating the endogenous locus, which provides the native chromosomal context with respect to DNA sequence and epigenetic milieu, and for proteins, [...], Saturation mutagenesis (1, 2)--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory (3) and protein-coding (4) nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or 'safe-harbour' integration fail to capture the native context of the endogenous chromosomal locus (5). This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/ Cas9 RNA-guided cleavage (6) with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.