RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Causes of Indonesia’s forest fires Edwards, Ryan B.; Naylor, Rosamond L.; Higgins, Matthew M. ...
World development,
03/2020, Letnik:
127
Journal Article
Recenzirano
•Redistricting increases fire and exacerbates the impacts El Nino on fire in Indonesia.•Regional economic growth has gone hand-in-hand with the use of fire in Indonesian rural districts.•Within ...districts, villages more likely to burn in 2015 tended to be more remote, less developed, and have a history of burning for agriculture.
The economic costs of Indonesia’s 2015 forest fires are estimated to exceed US $16 billion, with more than 100,000 premature deaths. On several days the fires emitted more carbon dioxide than the entire United States economy. Here, we combine detailed geospatial data on fire and local climatic conditions with rich administrative data to assess the underlying causes of Indonesia’s forest fires at district and village scales. We find that El Niño events explain most of the year-on-year variation in fire. The creation of new districts increases fire and exacerbates the El Niño impacts on fire. We also find that regional economic growth has gone hand-in-hand with the use of fire in rural districts. We proceed with a 30,000-village case study of the 2015 fire season on Sumatra and Kalimantan and ask which villages, for a given level of spatial fire risk, are more likely to have fire. Villages more likely to burn tend to be more remote, to be considerably less developed, and to have a history of using fire for agriculture. Although central and district level policies and regional economic development have generally contributed to voracious environmental degradation, the close link between poverty and fire at the village level suggests that the current policy push for village development might offer opportunities to reverse this trend.
Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, ...have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.
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•Genome-wide comparison of DNA replication timing between stem cell types•Aberrant replication timing in a subset of induced pluripotent stem cell lines•Delayed replication tends to occur near centromeres and telomeres•Aberrant replication timing is maintained following stem cell differentiation
Edwards et al. compare DNA replication timing between human embryonic stem cells and stem cells reprogrammed by defined factors or through nuclear transfer. Induced pluripotent stem cells incur aberrant replication timing at specific genomic regions in a subset of cell lines. These aberrations are carried through stem cell differentiation.
DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of ...replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.
Decentralization and the environment Naylor, Rosamond L.; Higgins, Matthew M.; Edwards, Ryan B. ...
Ambio,
10/2019, Letnik:
48, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Indonesia’s oil palm expansion during the last two decades has resulted in widespread environmental and health damages through land clearing by fire and peat conversion, but it has also contributed ...to rural poverty alleviation. In this paper, we examine the role that decentralization has played in the process of Indonesia’s oil palm development, particularly among independent smallholder producers. We use primary survey information, along with government documents and statistics, to analyze the institutional dynamics underpinning the sector’s impacts on economic development and the environment. Our analysis focuses on revenue-sharing agreements between district and central governments, district splitting, land title authority, and accountability at individual levels of government. We then assess the role of Indonesia’s Village Law of 2014 in promoting rural development and land clearing by fire. We conclude that both environmental conditionality and positive financial incentives are needed within the Village Law to enhance rural development while minimizing environmental damages.
Mutations in the
gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To ...identify the causal defect in AOA2, patient-derived cells and
knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to
mutation-associated chromosome fragility and AOA2.
Biological diversity depends on the interplay between evolutionary diversification and ecological mechanisms allowing species to coexist. Current research increasingly integrates ecology and ...evolution over a range of timescales, but our common conceptual framework for understanding species coexistence requires better incorporation of evolutionary processes. Here, we focus on the idea of evolutionarily stable communities (ESCs), which are theoretical endpoints of evolution in a community context. We use ESCs as a unifying framework to highlight some important but under‐appreciated theoretical results, and we review empirical research relevant to these theoretical predictions. We explain how, in addition to generating diversity, evolution can also limit diversity by reducing the effectiveness of coexistence mechanisms. The coevolving traits of competing species may either diverge or converge, depending on whether the number of species in the community is low (undersaturated) or high (oversaturated) relative to the ESC. Competition in oversaturated communities can lead to extinction or neutrally coexisting, ecologically equivalent species. It is critical to consider trait evolution when investigating fundamental ecological questions like the strength of different coexistence mechanisms, the feasibility of ecologically equivalent species, and the interpretation of different patterns of trait dispersion.
Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we ...profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.
Introduction
Individuals with sickle cell disease (SCD) at increased risk for stroke should undergo annual stroke risk assessment using transcranial Doppler (TCD) screening between the ages of 2 and ...16. Though this screening can significantly reduce morbidity associated with SCD, screening rates at Boston Children's Hospital (and nationwide) remain below the recommended 100% screening adherence rates.
Methods
Three plan–do–study–act (PDSA) cycles were designed and implemented. The Specific, Measurable, Achievable, Relevant, and Time‐Bound (SMART) aim of our quality improvement (QI) initiative was to sustainably increase the proportion of eligible patients receiving a TCD within 15 months of their last TCD to greater than 95%. An interrupted time series (ITS) analysis was performed, comparing TCD adherence rates from PDSA Cycle 1 to those from PDSA Cycles 2 and 3.
Results
Mean TCD adherence increased across all three PDSA cycles, from a baseline of 67% in the first cycle (January 2015 to September 2020) to 92% in the third cycle (May 2021 to March 2023). In the ITS analysis of TCD adherence rates, there was a significant difference in the final TCD adherence rate achieved compared to the rate predicted, with a total estimated increase in adherence of 17.9% being attributable to the interventions from PDSA Cycles 2 and 3.
Discussion
Although other QI initiatives had demonstrated ability to increase adherence to TCD screening for patients with SCD, this is the first QI project to collect data over such a prolonged period of time to demonstrate a sustained increase in screening rates throughout the intervention (an 8‐year period).
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