One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis.

The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2ß using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2ß-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2ß-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration.

Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase.

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.

Interpretable online network dictionary learning for inferring long-range chromatin interactions.

Dictionary learning (DL), implemented via matrix factorization (MF), is commonly used in computational biology to tackle ubiquitous clustering problems. The method is favored due to its conceptual simplicity and relatively low computational complexity. However, DL algorithms produce results that lack interpretability in terms of real biological data. Additionally, they are not optimized for graph-structured data and hence often fail to handle them in a scalable manner. In order to address these limitations, we propose a novel DL algorithm called online convex network dictionary learning (online cvxNDL). Unlike classical DL algorithms, online cvxNDL is implemented via MF and designed to handle extremely large datasets by virtue of its online nature. Importantly, it enables the interpretation of dictionary elements, which serve as cluster representatives, through convex combinations of real measurements. Moreover, the algorithm can be applied to data with a network structure by incorporating specialized subnetwork sampling techniques. To demonstrate the utility of our approach, we apply cvxNDL on 3D-genome RNAPII ChIA-Drop data with the goal of identifying important long-range interaction patterns (long-range dictionary elements). ChIA-Drop probes higher-order interactions, and produces data in the form of hypergraphs whose nodes represent genomic fragments. The hyperedges represent observed physical contacts. Our hypergraph model analysis has the objective of creating an interpretable dictionary of long-range interaction patterns that accurately represent global chromatin physical contact maps. Through the use of dictionary information, one can also associate the contact maps with RNA transcripts and infer cellular functions. To accomplish the task at hand, we focus on RNAPII-enriched ChIA-Drop data from Drosophila Melanogaster S2 cell lines. Our results offer two key insights. First, we demonstrate that online cvxNDL retains the accuracy of classical DL (MF) methods while simultaneously ensuring unique interpretability and scalability. Second, we identify distinct collections of proximal and distal interaction patterns involving chromatin elements shared by related processes across different chromosomes, as well as patterns unique to specific chromosomes. To associate the dictionary elements with biological properties of the corresponding chromatin regions, we employ Gene Ontology (GO) enrichment analysis and perform multiple RNA coexpression studies.

Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations.

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.

Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

Early systemic immune biomarkers predict bone regeneration after trauma.

Severe traumatic injuries are a widespread and challenging clinical problem, and yet the factors that drive successful healing and restoration of function are still not well understood. One recently identified risk factor for poor healing outcomes is a dysregulated immune response following injury. In a preclinical model of orthopedic trauma, we demonstrate that distinct systemic immune profiles are correlated with impaired bone regeneration. Most notably, elevated blood levels of myeloid-derived suppressor cells (MDSCs) and the immunosuppressive cytokine interleukin-10 (IL-10) are negatively correlated with functional bone regeneration as early as 1 wk posttreatment. Nonlinear multivariate regression also implicated these two factors as the most influential in predictive computational models. These results support a significant relationship between early systemic immune responses to trauma and subsequent local bone regeneration and indicate that elevated circulating levels of MDSCs and IL-10 may be predictive of poor functional healing outcomes and represent novel targets for immunotherapeutic intervention.

A Cross-Sectional Analysis of the Relationship Between Sabbath Practices and US, Canadian, Indonesian, and Paraguayan Teachers' Burnout.

Across different faith traditions, Sabbath day observance shares a close relationship with theological conceptions of rest. Sabbath-keeping, with its promise of rest, may be a valuable spiritual practice in the context of teaching as prior research has consistently documented the adverse effects of teacher burnout. Yet no research has examined Sabbath-keeping and its connections to teaching practices and teacher burnout. We aim to fill this gap with a quantitative study of Sabbath-keeping and burnout among 1,300 teachers in Christian schools throughout the USA, Canada, Indonesia, and Paraguay. We report their conceptions of Sabbath and how those conceptions inform their teaching practice. We find an inverse and statistically significant relationship between Sabbath-keeping and burnout that is robust across several model specifications, suggesting that Sabbath-keeping may be helpful in reducing burnout among educators.

Depletion of fat-resident Treg cells prevents age-associated insulin resistance.

Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult-onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR are largely unexplored. Here we show, using comparative adipo-immune profiling in mice, that fat-resident regulatory T cells, termed fTreg cells, accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying IR, mice deficient in fTreg cells are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. By contrast, selective depletion of fTreg cells via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie ageing- and obesity-associated IR, and implicate fTreg cells as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR.

C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma.

Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion C11orf95-RELA is an oncogenic driver in ST-EPN [Parker et al. Nature 506:451-455 (2014); Pietsch et al. Acta Neuropathol 127:609-611 (2014)] but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

Correction to: C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma.

In the original publication, Fig. 1 was incorrectly published with same two histograms at the bottom.

Intravenous Immunoglobulin and Methylprednisolone for Clozapine-Associated Perimyocarditis.

CLINICAL FEATURES: Cardiotoxicity is a rare but serious side effect of clozapine. We present a case of a psychiatric patient on chronic clozapine 75 mg daily, who presented with congestive heart failure secondary to the cardiotoxic effects of the psychiatric medication. THERAPEUTIC CHALLENGE: Conventional heart failure treatment failed to improve symptoms. SOLUTION: A course of 40 mg of intravenous immunoglobulin and 125 mg of steroids was implemented, after which the patient made a full recovery. We hope to raise awareness of concurrent clozapine-induced pericarditis and myocarditis and propose a role of intravenous immunoglobulin and steroids in the treatment of drug-induced cardiomyopathy.

A Delphi-Based Consensus Statement on the Management of Anticoagulated Patients With Botulinum Toxin for Limb Spasticity.

OBJECTIVE: To create a consensus statement on the considerations for treatment of anticoagulated patients with botulinum toxin A (BoNTA) intramuscular injections for limb spasticity. DESIGN: We used the Delphi method. SETTING: A multiquestion electronic survey. PARTICIPANTS: Canadian physicians (N=39) who use BoNTA injections for spasticity management in their practice. INTERVENTIONS: After the survey was sent, there were e-mail discussions to facilitate an understanding of the issues underlying the responses. Consensus for each question was reached when agreement level was ≥75%. MAIN OUTCOME MEASURES: Not applicable. RESULTS: When injecting BoNTA in anticoagulated patients: (1) BoNTA injections should not be withheld regardless of muscles injected; (2) a 25G or smaller size needle should be used when injecting into the deep leg compartment muscles; (3) international normalized ratio (INR) level should be ≤3.5 when injecting the deep leg compartment muscles; (4) if there are clinical concerns such as history of a fluctuating INR, recent bleeding, excessive or new bruising, then an INR value on the day of injection with point-of-care testing or within the preceding 2-3 days should be taken into consideration when injecting deep compartment muscles; (5) the concern regarding bleeding when using direct oral anticoagulants (DOACs) should be the same as with warfarin (when INR is in the therapeutic range); (6) the dose and scheduling of DOACs should not be altered for the purpose of minimizing the risk of bleeding prior to BoNTA injections. CONCLUSIONS: These consensus statements provide a framework for physicians to consider when injecting BoNTA for spasticity in anticoagulated patients. These consensus statements are not strict guidelines or decision-making steps, but rather an effort to generate common understanding in the absence of evidence in the literature.

Data Access Based on a Guide Map of the Underwater Wireless Sensor Network.

Underwater wireless sensor networks (UWSNs) represent an area of increasing research interest, as data storage, discovery, and query of UWSNs are always challenging issues. In this paper, a data access based on a guide map (DAGM) method is proposed for UWSNs. In DAGM, the metadata describes the abstracts of data content and the storage location. The center ring is composed of nodes according to the shortest average data query path in the network in order to store the metadata, and the data guide map organizes, diffuses and synchronizes the metadata in the center ring, providing the most time-saving and energy-efficient data query service for the user. For this method, firstly the data is stored in the UWSN. The storage node is determined, the data is transmitted from the sensor node (data generation source) to the storage node, and the metadata is generated for it. Then, the metadata is sent to the center ring node that is the nearest to the storage node and the data guide map organizes the metadata, diffusing and synchronizing it to the other center ring nodes. Finally, when there is query data in any user node, the data guide map will select a center ring node nearest to the user to process the query sentence, and based on the shortest transmission delay and lowest energy consumption, data transmission routing is generated according to the storage location abstract in the metadata. Hence, specific application data transmission from the storage node to the user is completed. The simulation results demonstrate that DAGM has advantages with respect to data access time and network energy consumption.

Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis.

The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation.

CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report.

OBJECTIVE: To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. APPROACH AND RESULTS: The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. CONCLUSIONS: CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.

SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state.

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.

CRISPR-Cas9-mediated genome editing and guide RNA design.

CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR-Cas9 system holds promise as a "system of choice" for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRISPR-Cas9 system and its application to genome editing, with a focus on the basic considerations in designing the targeting guide RNA sequence.