Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or ...deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
Many researchers have focused on the investigation into frost formation mechanism and tried to find various effective defrosting methods in recent years. Study on frost is divided into three stages: ...the frost formation process and mechanism, defrosting methods, and restraint frost methods. The three stages are carried out sequentially or in parallel. Compared to defrost, restraint frost is becoming more welcomed by peoples because of no energy or smaller consume. The affecting factors on frosting directly induce frost formation on cold surface, which leads to blockage, performance decrease, and even malfunction of the low temperature heat exchanger. This paper divides the factors into three categories: the characteristics (temperature, humidity and velocity) of the moist air, the features （temperature, structure and position, treatment）of the cold surfaces, the interaction between the air or the formed frost and the cold surface (electric field, ultrasonic wave, magnetic field, oscillation effects). One or several of the factors must be changed in order to restrain frost formation. Relative humidity is considered as that it has a larger effect on the frost formation, compared to the air temperature and air velocity. However, only a few researches pay attention to the restraint frost by controlling the characteristics of moist air. The structural parameters such as the fin spaces have the most important effect on the heat transfer performance of a heat exchanger under frosting conditions. Therefore, an optimization of the heat exchanger design should be considered for restraint frost. Hydrophobic surface is recognized as an effective way to improve the energy efficiency of a refrigeration system under frosting conditions. However, the research results are not as satisfactory as expected because of the difficult fabrication of scalable hydrophobic properties.
Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal ...hemoglobin (HbF, α
γ
)
. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells
. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
We previously identified that the development of early-stage myeloid-derived suppressor cells (eMDSCs) in breast cancer with high IL-6 (IL-6
) expression was correlated with the SOCS3 ...deficiency-dependent hyperactivation of the JAK/STAT signaling pathway. However, the regulatory mechanisms have not yet been elucidated. In this study, we aimed to investigate how the posttranscriptional regulation mediated by cancer exosome-derived miRNAs affected the JAK/STAT signaling pathway and the development of eMDSCs. Using miRNA microarray, we screened miR-9 and miR-181a which were exclusively upregulated in eMDSCs and inversely associated with SOCS3 expression. We found both miRNAs promoted the amplification of immature eMDSCs with the strong suppression on T-cell immunity in mice and humans. Furthermore, miR-9 and miR-181a promoted 4T1 tumor growth and immune escape via enhancing eMDSCs infiltration in situ. But miR-9 and miR-181a stimulated eMDSCs development by separately inhibiting SOCS3 and PIAS3, two crucial regulators in the negative feedback loop of the JAK/STAT signaling pathway. Elevated miR-9 and miR-181a in eMDSCs was derived from tumor-derived exosomes, and blocking the exosome release could fully attenuate the miRNA-mediated regulation on eMDSCs development. In summary, our findings indicated that tumor exosome-derived miR-9 and miR-181a activated the JAK/STAT signaling pathway via targeting SOCS3 and PIAS3, respectively, and thus promoted the expansion of eMDSCs which might provide potential therapeutic target for IL-6
breast cancer treatment.
Abstract As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. ...The mitochondrial unfolded protein response (UPR mt ) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPR mt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPR mt activation.
Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress ...protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H
S) production. This H
S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H
S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H
S levels, or enhancing mechanisms that H
S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.
The lncRNA SNHG6 (small nucleolar RNA host gene 6) plays vital roles in tumorigenesis and the progression of hepatocellular carcinoma (HCC). However, the regulatory mechanisms of SNHG6 are largely ...unknown. In this study, we identified, via quantitative proteomics, specific cytoskeleton-associated proteins and enzyme modulators to be potential targets of SNHG6. SNHG6 reduced the mRNA levels of lysine methyltransferase, SET domain containing 7 (SETD7) and leucine zipper transcription factor-like 1 (LZTFL1) by posttranscriptional destabilization. Silencing of SETD7 or LZTFL1 reversed the suppressive effects of SNHG6 knockdown on HCC progression. Heterogeneous nuclear ribonucleoprotein L (HNRNPL) and polypyrimidine tract binding protein 1 (PTBP1) were identified as SNHG6-interacting proteins that bind to SETD7 or LZTFL1 mRNA. Forced expression of SNHG6 led to HNRNPL being competitively adsorbed by SNHG6, thereby removing its stabilizing effect on SETD7. Concurrently, the functional SNHG6-PTBP1 complex facilitated the degradation of LZTFL1 mRNA in hepatoma cells. These results indicated that SNHG6 promotes HCC progression by functioning as a “decoy plus guide” for HNRNPL and PTBP1 to facilitate mRNA decay of SETD7 and LZTFL1, thereby serving as a novel therapeutic target for HCC.
•SNHG6 reduces SETD7 and LZTFL1 mRNA levels by post-transcriptional destabilization.•SNHG6-interacting proteins include RNA-binding proteins HNRNPL and PTBP1.•HNRNPL is competitively adsorbed by SNHG6, destabilizing SETD7 mRNA.•The functional SNHG6-PTBP1 complex facilitates the degradation of LZTFL1 mRNA.•SNHG6 promotes HCC progression by functioning as a “decoy plus guide”.
Energy-efficient computation is important in mobile edge computing (MEC) systems. However, the computation efficiency problem in the unmanned aerial vehicle (UAV)-enabled MEC systems has been rarely ...researched. In this paper, a UAV-enabled MEC system under partial computation offloading mode is investigated. The computation efficiency is maximized by jointly optimizing the offloading times, the central processing unit frequencies, the transmit powers of the user and the trajectory of the UAV. For the non-convex computation efficiency problem, a two-stage iterative algorithm is proposed. Moreover, we derive the closed-form expressions for the local computation frequency and the transmit power of the user, which facilitates our algorithm implementation. Simulation results demonstrate that the computation efficiency of our proposed joint optimization scheme is better than those of other benchmark schemes.
CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many ...hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an
transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an
transgene bearing miRNA-binding sites in its 3'-UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3
construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.
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