PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.

The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.

FOXO3a-interacting proteins' involvement in cancer: a review.

Due to its role in apoptosis, differentiation, cell cycle arrest, and DNA damage repair in stress responses (oxidative stress, hypoxia, chemotherapeutic drugs, and UV irradiation or radiotherapy), FOXO3a is considered a key tumor suppressor that determines radiotherapeutic and chemotherapeutic responses in cancer cells. Mutations in the FOXO3a gene are rare, even in cancer cells. Post-translational regulations are the main mechanisms for inactivating FOXO3a. The subcellular localization, stability, transcriptional activity, and DNA binding affinity for FOXO3a can be modulated via various post-translational modifications, including phosphorylation, acetylation, and interactions with other transcriptional factors or regulators. This review summarizes how proteins that interact with FOXO3a engage in cancer progression.

Fluorescent probes for ferroptosis bioimaging: advances, challenges, and prospects.

Ferroptosis is a form of regulatory cell death distinct from caspase-dependent apoptosis and plays an important role in life entities. Since ferroptosis involves a variety of complex regulatory factors, the levels of certain biological species and microenvironments would change during this process. Thus, the investigation of the level fluctuation of key target analytes during ferroptosis is of great significance for disease treatment and drug design. Toward this aim, multiple organic fluorescent probes with simple preparation and non-destructive detection have been developed, and research over the past decade has uncovered a vast array of homeostasis and other physiological characteristics of ferroptosis. However, this significant and cutting-edge topic has not yet been reviewed. In this work, we aim to highlight the latest breakthrough results of fluorescent probes for monitoring various bio-related molecules and microenvironments during ferroptosis at the cellular, tissue and in vivo levels. Accordingly, this tutorial review has been organized according to the target molecules identified by the probes including ionic species, reactive sulfur species, reactive oxygen species, biomacromolecules, microenvironment, and others. In addition to providing new insights into the findings of each fluorescent probe in ferroptosis studies, we also discuss the defects and limitations of the probes developed, and highlight the potential challenges and further prospects in this domain. We anticipate that this review will convey profound implications for designing powerful fluorescent probes to decrypt changes in key molecules and microenvironments during ferroptosis.

Investigating the Therapeutic Effects of Ferroptosis on Myocardial Ischemia-Reperfusion Injury Using a Dual-Locking Mitochondrial Targeting Strategy.

Research on ferroptosis in myocardial ischemia/reperfusion injury (MIRI) using mitochondrial viscosity as a nexus holds great promise for MIRI therapy. However, high-precision visualisation of mitochondrial viscosity remains a formidable task owing to the debilitating electrostatic interactions caused by damaged mitochondrial membrane potential. Herein, we propose a dual-locking mitochondria-targeting strategy that incorporates electrostatic forces and probe-protein molecular docking. Even in damaged mitochondria, stable and precise visualisation of mitochondrial viscosity in triggered and medicated MIRI was achieved owing to the sustained driving forces (e.g., pi-cation, pi-alkyl interactions, etc.) between the developed probe, CBS, and the mitochondrial membrane protein. Moreover, complemented by a western blot, we confirmed that ferrostatin-1 exerts its therapeutic effect on MIRI by improving the system xc-/GSH/GPX4 antioxidant system, confirming the therapeutic value of ferroptosis in MIRI. This study presents a novel strategy for developing robust mitochondrial probes, thereby advancing MIRI treatment.

Brain-derived neurotrophic factor regulates LYN kinase-mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons.

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing.

BACKGROUND: Rewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the entire biological field. Since its emergence, this technology has been widely applied to induce gene knockouts, insertions, deletions, and base substitutions. However, the classical version of this system was imperfect for inducing or correcting desired mutations. A subsequent development generated more advanced classes, including cytosine and adenine base editors, which can be used to achieve single nucleotide substitutions. Nevertheless, these advanced systems still suffer from several limitations, such as the inability to edit loci without a suitable PAM sequence and to induce base transversions. On the other hand, the recently emerged prime editors (PEs) can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions, which show promising potential to alter and correct the genomes of various organisms. Of note, the application of PE to edit livestock genomes has not been reported yet. RESULTS: In this study, using PE, we successfully generated sheep with two agriculturally significant mutations, including the fecundity-related FecBB p.Q249R and the tail length-related TBXT p.G112W. Additionally, we applied PE to generate porcine blastocysts with a biomedically relevant point mutation (KCNJ5 p.G151R) as a porcine model of human primary aldosteronism. CONCLUSIONS: Our study demonstrates the potential of the PE system to edit the genomes of large animals for the induction of economically desired mutations and for modeling human diseases. Although prime-edited sheep and porcine blastocysts could be generated, the editing frequencies are still unsatisfactory, highlighting the need for optimizations in the PE system for efficient generation of large animals with customized traits.

Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats.

Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high > 3000 m above sea level and low < 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (PGWAS = 3.62 × 10-25) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection.

SERPINB8 and furin regulate ITGAX expression and affect the proliferation and invasion of melanoma cells.

In the past 10 years, the systemic treatment of advanced melanoma has undergone tremendous changes through the development of targeted therapy. However, there is still a long way to go. This study aims to characterize the function and interaction of ITGAX, SERPINB8 and furin in BRAF V600E mutant melanoma. Differentially expressed genes related to BRAF V600E mutation and BRAFi treatment were obtained by analysing GSE141484 and GSE22838. two kinds of BRAFi (Vemurafenib, 10 µM; Dabrafenib, 1 µM) were used to treat A375 and 1205Lu cells, respectively. The expression of ITGAX, SERPINB8 and Furin in A375 and 1205Lu cells was down-regulated by specific siRNAs, and cell proliferation, clone formation and invasion were detected by CCK-8, colony formation and transwell assays. The physical binding of furin and SERPINB8 was detected by immunoprecipitation. BRAFi treatment down-regulated the ITGAX and SERPINB8 expression and did not change furin expression. Knockdown of ITGAX and SERPINB8 both inhibited the proliferation and invasion of A375 and 1205Lu cells. Knocking down SERPINB8 down-regulated the expression of ITGAX. Furin knockdown and inhibitors all up-regulated the protein level of ITGAX. SERPINB8 can physically bind to furin. In summary, SERPINB8 and furin regulate the expression of ITGAX in melanoma cells, and ITGAX significantly promotes the proliferation and invasion of melanoma cells.

Bimetallic Organic Framework-Decorated Leaf-like 2D Nanosheets as Flexible Air Cathode for Rechargeable Zn-air Batteries.

Exploring highly active and robust self-supporting air electrodes is the key for flexible Zn-air batteries (FZABs). Therefore, we report a novel 3D structural bimetal-based self-supporting electrode consisting of hybrid Cu, Co nanoparticles co-modified nitrogen-doped carbon nanosheets on carbon cloth (Cu, Co NPs@NCNSs/CC), which displays excellent electrochemical activity and durability of the oxygen reduction/evolution reaction (ORR/OER). The Cu, Co NPs@NCNSs/CC exhibits a half-wave potential of 0.863 V toward ORR and an overpotential of 225 mV at 10 mA cm-2 toward OER, owing to its exposed bimetallic sites accelerating the kinetic reaction. In addition, the density functional theory calculation proves that the synergistic effect of CuCo sites favors ORR and OER. Hence, the FZABs based on Cu, Co NPs@NCNSs/CC achieve a larger open-circuit potential (1.45 V), higher energy density (130.10 mW cm-2 ), and outstanding cycling stability. All remarkable results demonstrate valuable enlightenment for seeking advanced energy materials of portable and wearable electronics.

Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.

BACKGROUND: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. RESULTS: We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/µL Cas9 mRNA and 200 ng/µL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. CONCLUSIONS: This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.