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1.
Nat Commun ; 10(1): 4063, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492858

RESUMO

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

2.
Neuron ; 103(3): 412-422.e4, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31221560

RESUMO

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.

3.
Nat Genet ; 51(4): 592-599, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30926968

RESUMO

Transcriptome-wide association studies (TWAS) integrate genome-wide association studies (GWAS) and gene expression datasets to identify gene-trait associations. In this Perspective, we explore properties of TWAS as a potential approach to prioritize causal genes at GWAS loci, by using simulations and case studies of literature-curated candidate causal genes for schizophrenia, low-density-lipoprotein cholesterol and Crohn's disease. We explore risk loci where TWAS accurately prioritizes the likely causal gene as well as loci where TWAS prioritizes multiple genes, some likely to be non-causal, owing to sharing of expression quantitative trait loci (eQTL). TWAS is especially prone to spurious prioritization with expression data from non-trait-related tissues or cell types, owing to substantial cross-cell-type variation in expression levels and eQTL strengths. Nonetheless, TWAS prioritizes candidate causal genes more accurately than simple baselines. We suggest best practices for causal-gene prioritization with TWAS and discuss future opportunities for improvement. Our results showcase the strengths and limitations of using eQTL datasets to determine causal genes at GWAS loci.


Assuntos
Predisposição Genética para Doença/genética , Transcriptoma/genética , Doença de Crohn/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Lipoproteínas LDL/genética , Locos de Características Quantitativas/genética , Esquizofrenia/genética
4.
Nat Genet ; 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30510241

RESUMO

To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P < 5 × 10-8, bringing the number of known independent signals for CRC to ~100. New signals implicate lower-frequency variants, Krüppel-like factors, Hedgehog signaling, Hippo-YAP signaling, long noncoding RNAs and somatic drivers, and support a role for immune function. Heritability analyses suggest that CRC risk is highly polygenic, and larger, more comprehensive studies enabling rare variant analysis will improve understanding of biology underlying this risk and influence personalized screening strategies and drug development.

5.
Nat Biotechnol ; 36(9): 829-838, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30188539

RESUMO

Deep learning is beginning to impact biological research and biomedical applications as a result of its ability to integrate vast datasets, learn arbitrarily complex relationships and incorporate existing knowledge. Already, deep learning models can predict, with varying degrees of success, how genetic variation alters cellular processes involved in pathogenesis, which small molecules will modulate the activity of therapeutically relevant proteins, and whether radiographic images are indicative of disease. However, the flexibility of deep learning creates new challenges in guaranteeing the performance of deployed systems and in establishing trust with stakeholders, clinicians and regulators, who require a rationale for decision making. We argue that these challenges will be overcome using the same flexibility that created them; for example, by training deep models so that they can output a rationale for their predictions. Significant research in this direction will be needed to realize the full potential of deep learning in biomedicine.

6.
Genetics ; 206(4): 1727-1738, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28634160

RESUMO

Interactions between Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs and CRISPR-associated (Cas) proteins form an RNA-guided adaptive immune system in prokaryotes. The adaptive immune system utilizes segments of the genetic material of invasive foreign elements in the CRISPR locus. The loci are transcribed and processed to produce small CRISPR RNAs (crRNAs), with degradation of invading genetic material directed by a combination of complementarity between RNA and DNA and in some cases recognition of adjacent motifs called PAMs (Protospacer Adjacent Motifs). Here we describe a general, high-throughput procedure to test the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system. These studies were followed with reciprocal experiments in which the consequence of CRISPR activity was survival in the presence of a lytic phage. From the combined analysis of the Cascade system, we found that (i) type I-E Cascade PAM recognition is more expansive than previously reported, with at least 22 distinct PAMs, with many of the noncanonical PAMs having CRISPR-interference abilities similar to the canonical PAMs; (ii) PAM positioning appears precise, with no evidence for tolerance to PAM slippage in interference; and (iii) while increased guanine-cytosine (GC) content in the spacer is associated with higher CRISPR-interference efficiency, high GC content (>62.5%) decreases CRISPR-interference efficiency. Our findings provide a comprehensive functional profile of Cascade type I-E interference requirements and a method to assay spacer efficacy that can be applied to other CRISPR-Cas systems.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Polimorfismo Genético , RNA Guia/genética , Proteínas Associadas a CRISPR/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , RNA Guia/metabolismo
7.
Nat Commun ; 8: 15178, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28474669

RESUMO

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.


Assuntos
Sistemas CRISPR-Cas/genética , Marcação de Genes/métodos , Biblioteca Genômica , Ensaios de Triagem em Larga Escala/métodos , RNA Guia/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dano ao DNA/genética , Humanos , Polissacarídeos/biossíntese , Interferência de RNA , Ricina/toxicidade
8.
Hum Mutat ; 38(9): 1240-1250, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28220625

RESUMO

In many human diseases, associated genetic changes tend to occur within noncoding regions, whose effect might be related to transcriptional control. A central goal in human genetics is to understand the function of such noncoding regions: given a region that is statistically associated with changes in gene expression (expression quantitative trait locus [eQTL]), does it in fact play a regulatory role? And if so, how is this role "coded" in its sequence? These questions were the subject of the Critical Assessment of Genome Interpretation eQTL challenge. Participants were given a set of sequences that flank eQTLs in humans and were asked to predict whether these are capable of regulating transcription (as evaluated by massively parallel reporter assays), and whether this capability changes between alternative alleles. Here, we report lessons learned from this community effort. By inspecting predictive properties in isolation, and conducting meta-analysis over the competing methods, we find that using chromatin accessibility and transcription factor binding as features in an ensemble of classifiers or regression models leads to the most accurate results. We then characterize the loci that are harder to predict, putting the spotlight on areas of weakness, which we expect to be the subject of future studies.


Assuntos
Biologia Computacional/métodos , Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Locos de Características Quantitativas
9.
BMC Genomics ; 17(1): 787, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27717327

RESUMO

BACKGROUND: Alternative mRNA splicing is critical to proteomic diversity and tissue and species differentiation. Exclusion of cassette exons, also called exon skipping, is the most common type of alternative splicing in mammals. RESULTS: We present a computational model that predicts absolute (though not tissue-differential) percent-spliced-in of cassette exons more accurately than previous models, despite not using any 'hand-crafted' biological features such as motif counts. We achieve nearly identical performance using only the conservation score (mammalian phastCons) of each splice junction normalized by average conservation over 100 bp of the corresponding flanking intron, demonstrating that conservation is an unexpectedly powerful indicator of alternative splicing patterns. Using this method, we provide evidence that intronic splicing regulation occurs predominantly within 100 bp of the alternative splice sites and that conserved elements in this region are, as expected, functioning as splicing regulators. We show that among conserved cassette exons, increased conservation of flanking introns is associated with reduced inclusion. We also propose a new definition of intronic splicing regulatory elements (ISREs) that is independent of conservation, and show that most ISREs do not match known binding sites or splicing factors despite being predictive of percent-spliced-in. CONCLUSIONS: These findings suggest that one mechanism for the evolutionary transition from constitutive to alternative splicing is the emergence of cis-acting splicing inhibitors. The association of our ISREs with differences in splicing suggests the existence of novel RNA-binding proteins and/or novel splicing roles for known RNA-binding proteins.


Assuntos
Processamento Alternativo , Evolução Molecular , Modelos Biológicos , Animais , Área Sob a Curva , Encéfalo/metabolismo , Éxons , Regulação da Expressão Gênica , Humanos , Íntrons , Especificidade de Órgãos/genética , Sítios de Splice de RNA , Sequências Reguladoras de Ácido Nucleico
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