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.
CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells
. Whereas some cell types are amenable to genome engineering, genomes of human ...pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells
. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction
. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations
, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.
Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the ...receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.
The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose ...and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function—all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell’s metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.
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► Glucose and glutamine feed back to promote mTORC1 signaling through ATP production ► Energetic stress prevents mTOR lysosomal localization independently of AMPK and Rag ► ATP-dependent TTT-RUVBL complex is disassembled and repressed by energetic stress ► TTT-RUVBL is required for mTORC1 functional assembly and lysosomal localization
Friend leukemia virus integration 1 (FLI1), an ETS transcription factor family member, acts as an oncogenic driver in hematological malignancies and promotes tumor growth in solid tumors. However, ...little is known about the mechanisms underlying the activation of this proto-oncogene in tumors.
Immunohistochemical staining showed that FLI1 is aberrantly overexpressed in advanced stage and metastatic breast cancers. Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. Notably, FECR1 utilizes a positive feedback mechanism to activate FLI1 by inducing DNA hypomethylation in CpG islands of the promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1, a demethylase that is actively involved in DNA demethylation. FECR1 also binds to and downregulates in trans DNMT1, a methyltransferase that is essential for the maintenance of DNA methylation.
These data suggest that FECR1 circular RNA acts as an upstream regulator to control breast cancer tumor growth by coordinating the regulation of DNA methylating and demethylating enzymes. Thus, FLI1 drives tumor metastasis not only through the canonical oncoprotein pathway, but also by using epigenetic mechanisms mediated by its exonic circular RNA.
β-alanine is a nonessential amino acid that combines with the amino acid histidine to form the intracellular dipeptide carnosine, an important intracellular buffer. Evidence has been well established ...on the ability of β-alanine supplementation to enhance anaerobic skeletal muscle performance. As a result, β-alanine has become one of the more popular supplements used by competitive athletes. These same benefits have also been reported in soldiers. Evidence accumulated over the last few years has suggested that β-alanine can result in carnosine elevations in the brain, which appears to have broadened the potential effects that β-alanine supplementation may have on soldier performance and health. Evidence suggests that β-alanine supplementation can increase resilience to post-traumatic stress disorder, mild traumatic brain injury and heat stress. The evidence regarding cognitive function is inconclusive but may be more of a function of the stressor that is applied during the assessment period. The potential benefits of β-alanine supplementation on soldier resiliency are interesting but require additional research using a human model. The purpose of this review is to provide an overview of the physiological role of β-alanine and why this nutrient may enhance soldier performance.
Although they are but a small fraction of the mass ejected in core-collapse supernovae, neutrino-driven winds (NDWs) from nascent proto-neutron stars (PNSs) have the potential to contribute ...significantly to supernova nucleosynthesis. In previous works, the NDW has been implicated as a possible source of r-process and light p-process isotopes. In this paper, we present time-dependent hydrodynamic calculations of nucleosynthesis in the NDW which include accurate weak interaction physics coupled to a full nuclear reaction network. Using two published models of PNS neutrino luminosities, we predict the contribution of the NDW to the integrated nucleosynthetic yield of the entire supernova. For the neutrino luminosity histories considered, no true r-process occurs in the most basic scenario. The wind driven from an older 1.4 M{sub sun} model for a PNS is moderately neutron-rich at late times however, and produces {sup 87}Rb, {sup 88}Sr, {sup 89}Y, and {sup 90}Zr in near solar proportions relative to oxygen. The wind from a more recently studied 1.27 M{sub sun} PNS is proton-rich throughout its entire evolution and does not contribute significantly to the abundance of any element. It thus seems very unlikely that the simplest model of the NDW can produce the r-process. At most, it contributes to the production of the N = 50 closed shell elements and some light p-nuclei. In doing so, it may have left a distinctive signature on the abundances in metal-poor stars, but the results are sensitive to both uncertain models for the explosion and the masses of the neutron stars involved.
Objectives/Hypothesis
Current treatment options for idiopathic subglottic stenosis include endoscopic interventions, resection, and tracheotomy. Recently, serial office‐based steroid injections were ...proposed as an alternative that may stabilize or induce regression of airway stenosis without the need for repeated operations. Procedure completion rate, pain, complications, effect on stenosis, time since the last operation, and limitations have not been described.
Study Design
Retrospective case series.
Methods
Retrospective series of 19 patients undergoing serial office‐based steroid injection for idiopathic subglottic stenosis. Outcome measures included completion rate, procedure‐related pain scores, complications, percentage of airway stenosis, and time since the last operative intervention.
Results
Procedure completion rate was 98.8%. Average pain score during the procedure was 2.3 ± 1.7 on a 10‐point scale. There were no immediate complications. One patient underwent awake tracheotomy 8 days after her second injection and was later decannulated. Average stenosis decreased from 35% ± 15% to 25% ± 15% (n = 16; P = .086) over the first of three injections and 40% ± 15% to 25% ± 10% to 20% ± 10% (n = 8; P = .002) for those patients completing two sets of three injections. Fourteen of 17 patients undergoing at least three injections have not returned to the operating room since the first injection.
Conclusions
Office‐based steroid injection represents a promising new treatment pathway for a disease that requires long‐term management, offering a purely pharmacologic approach to a disorder that has traditionally been approached from a mechanical perspective. It is safe, well tolerated, and effective. Furthermore, it may help patients and physicians avoid repeated trips to the operating room and the associated risks.
Level of Evidence
4. Laryngoscope, 127:2475–2481, 2017
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