The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption ...of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using “error-prone” DNA-repair machinery to generate precise edits.
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•DNA repair profiles of 223 sites in the human genome reveal nonrandom outcomes•The protospacer sequence, not genomic context, determines Cas9 DSB repair outcomes•Each DNA repair profile is composed of specific contributions from c-NHEJ and MMEJ•DNA repair profiling can be used to anticipate repair outcomes in primary cells
van Overbeek, Capurso et al. demonstrate that repair outcomes are nonrandom at S. pyogenes Cas9-mediated DSBs and are determined by the protospacer sequence rather than genomic context. DNA repair profiling reveals specific contributions of c-NHEJ and MMEJ at each site and an approach to generate precise edits without exogenous template.
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.
Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show ...that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.
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•B-lymphoid cancer cells can escape to venetoclax by overexpressing MCL-1•Modulation of AMPK/PKA axis and lymphoid transcription drive venetoclax resistance•Venetoclax resistance involves changes in cellular energy metabolism such as OXPHOS•Metabolic modulators can cooperate with venetoclax to overcome resistance
Guièze et al. show that resistance to the BCL-2 inhibitor venetoclax in chronic lymphocytic leukemia is associated with complex clonal shifts and identify, in addition to the known involvement by BCL-2 family members, regulators of lymphoid transcription and cellular energy metabolism as resistance drivers.
ABSTRACT
In the seconds following their formation in core-collapse supernovae, ‘proto’-magnetars drive neutrino-heated magnetocentrifugal winds. Using a suite of two-dimensional axisymmetric ...magnetohydrodynamic simulations, we show that relatively slowly rotating magnetars with initial spin periods of P⋆0 = 50–500 ms spin down rapidly during the neutrino Kelvin–Helmholtz cooling epoch. These initial spin periods are representative of those inferred for normal Galactic pulsars, and much slower than those invoked for gamma-ray bursts and superluminous supernovae. Since the flow is non-relativistic at early times, and because the Alfvén radius is much larger than the proto-magnetar radius, spin-down is millions of times more efficient than the typically used dipole formula. Quasi-periodic plasmoid ejections from the closed zone enhance spin-down. For polar magnetic field strengths B0 ≳ 5 × 1014 G, the spin-down time-scale can be shorter than the Kelvin–Helmholtz time-scale. For B0 ≳ 1015 G, it is of the order of seconds in early phases. We compute the spin evolution for cooling proto-magnetars as a function of B0, P⋆0, and mass (M). Proto-magnetars born with B0 greater than $\simeq 1.3\times 10^{15}\, {\rm \, G}\, (P_{\star 0}/{400\, \rm \, ms})^{-1.4}(M/1.4\, {\rm M}_\odot)^{2.2}$ spin down to periods >1 s in just the first few seconds of evolution, well before the end of the cooling epoch and the onset of classic dipole spin-down. Spin-down is more efficient for lower M and for larger P⋆0. We discuss the implications for observed magnetars, including the discrepancy between their characteristic ages and supernova remnant ages. Finally, we speculate on the origin of 1E 161348−5055 in the remnant RCW 103, and the potential for other ultra-slowly rotating magnetars.
Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating ...oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases
. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo
. Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.
ABSTRACT
Rapidly rotating magnetars have been associated with gamma-ray bursts (GRBs) and superluminous supernovae (SLSNe). Using a suite of two-dimensional magnetohydrodynamic simulations at fixed ...neutrino luminosity and a couple of evolutionary models with evolving neutrino luminosity and magnetar spin period, we show that magnetars are viable central engines for powering GRBs and SLSNe. We also present analytical estimates of the energy outflow rate from the proto-neutron star (PNS) as a function of polar magnetic field strength B0, PNS angular velocity Ω⋆, PNS radius R⋆, and mass outflow rate $\dot{M}$. We show that rapidly rotating magnetars with spin periods P⋆ ≲ 4 ms and polar magnetic field strength B0 ≳ 1015 G can release 1050 to 5 × 1051 erg of energy during the first ∼2 s of the cooling phase. Based on this result, it is plausible that sustained energy injection by magnetars through the relativistic wind phase can power GRBs. We also show that magnetars with moderate field strengths of B0 ≲ 5 × 1014 G do not release a large fraction of their rotational kinetic energy during the cooling phase and, hence, are not likely to power GRBs. Although we cannot simulate to times greater than ∼3–5 s after a supernova, we can hypothesize that moderate field strength magnetars can brighten the supernova light curves by releasing their rotational kinetic energy via magnetic dipole radiation on time-scales of days to weeks, since these do not expend most of their rotational kinetic energy during the early cooling phase.
There is a growing realization that early life influences have lasting impact on brain function and structure. Recent research has demonstrated that genetic relationships in adults can be used to ...parcellate the cortex into regions of maximal shared genetic influence, and a major hypothesis is that genetically programmed neurodevelopmental events cause a lasting impact on the organization of the cerebral cortex observable decades later. Here we tested how developmental and lifespan changes in cortical thickness fit the underlying genetic organizational principles of cortical thickness in a longitudinal sample of 974 participants between 4.1 and 88.5 y of age with a total of 1,633 scans, including 773 scans from children below 12 y. Genetic clustering of cortical thickness was based on an independent dataset of 406 adult twins. Developmental and adult age-related changes in cortical thickness followed closely the genetic organization of the cerebral cortex, with change rates varying as a function of genetic similarity between regions. Cortical regions with overlapping genetic architecture showed correlated developmental and adult age change trajectories and vice versa for regions with low genetic overlap. Thus, effects of genes on regional variations in cortical thickness in middle age can be traced to regional differences in neurodevelopmental change rates and extrapolated to further adult aging-related cortical thinning. This finding suggests that genetic factors contribute to cortical changes through life and calls for a lifespan perspective in research aimed at identifying the genetic and environmental determinants of cortical development and aging.
Life in the ocean will increasingly have to contend with a complex matrix of concurrent shifts in environmental properties that impact their physiology and control their life histories. Rhodoliths ...are coralline red algae (Corallinales, Rhodophyta) that are photosynthesizers, calcifiers, and ecosystem engineers and therefore represent important targets for ocean acidification (OA) research. Here, we exposed live rhodoliths to near-future OA conditions to investigate responses in their photosynthetic capacity, calcium carbonate production, and associated microbiome using carbon uptake, decalcification assays, and whole genome shotgun sequencing metagenomic analysis, respectively. The results from our live rhodolith assays were compared to similar manipulations on dead rhodolith (calcareous skeleton) biofilms and water column microbial communities, thereby enabling the assessment of host-microbiome interaction under climate-driven environmental perturbations.
Under high pCO
conditions, live rhodoliths exhibited positive physiological responses, i.e. increased photosynthetic activity, and no calcium carbonate biomass loss over time. Further, whereas the microbiome associated with live rhodoliths remained stable and resembled a healthy holobiont, the microbial community associated with the water column changed after exposure to elevated pCO
.
Our results suggest that a tightly regulated microbial-host interaction, as evidenced by the stability of the rhodolith microbiome recorded here under OA-like conditions, is important for host resilience to environmental stress. This study extends the scarce comprehension of microbes associated with rhodolith beds and their reaction to increased pCO
, providing a more comprehensive approach to OA studies by assessing the host holobiont.
Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause ...marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.
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