The efficiency of homology-directed repair (HDR) plays a crucial role in the development of animal models and gene therapy. We demonstrate that microhomology-mediated end-joining (MMEJ) constitutes a ...substantial proportion of DNA repair during CRISPR-mediated gene editing. Using CasRx to downregulate a key MMEJ factor, Polymerase Q (Polq), we improve the targeted integration efficiency of linearized DNA fragments and single-strand oligonucleotides (ssODN) in mouse embryos and offspring. CasRX-assisted targeted integration (CATI) also leads to substantial improvements in HDR efficiency during the CRISPR/Cas9 editing of monkey embryos. We present a promising tool for generating monkey models and developing gene therapies for clinical trials.
Base editing installs a precise nucleotide change in specific gene loci without causing a double-strand break. Its efficiency in human embryos is generally low, limiting its utility in functional ...genetic studies. Here, we report that injecting base editors into human cleaving two-cell and four-cell embryos results in much higher (up to 13-fold) homozygotic nucleotide substitution efficiency as opposed to MII oocytes or zygotes. Furthermore, as a proof-of-principle study, a point mutation can be efficiently corrected by our method. Our study indicates that human cleaving embryos provide an efficient base editing window for robust gene disruption and correction.
Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information ...are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.
Abstract
N
6
-methyladenosine (m
6
A) modification has been shown to regulate RNA metabolism. Here, we investigate m
6
A dynamics during maternal-to-zygotic transition (MZT) in mice through ...multi-omic analysis. Our results show that m
6
A can be maternally inherited or de novo gained after fertilization. Interestingly, m
6
A modification on maternal mRNAs not only correlates with mRNA degradation, but also maintains the stability of a small group of mRNAs thereby promoting their translation after fertilization. We identify Ythdc1 and Ythdf2 as key m
6
A readers for mouse preimplantation development. Our study reveals a key role of m
6
A mediated RNA metabolism during MZT in mammals.
Drip irrigation is an effective method to utilize waste saline-sodic land with a high water table. For reasonable and sustainable utilization of saline-sodic soil under such conditions, ...spatiotemporal changes in total nitrogen (TN), total phosphorus (TP), and soil organic matter (SOM) were investigated during the utilization process. The soil was sampled from newly built raised beds before planting (0 y) and beds in three adjacent plots had been planted with Lycium barbarum L. for one (1 y), two (2 y), and three years (3 y), respectively, at the end of the growing season. Soil samples were obtained at four horizontal distances from the drip line (0, 10, 20, and 30 cm) and four vertical soil depths (0–10, 10–20, 20–30, and 30–40 cm). The results showed that the average TN and TP of the soil profile increased with the planting year and were approximately 0.68 and 1.81 g·kg−1 in the soils of 3 y, approximately 84.9 and 42.4% higher than that of 0 y, respectively. SOM decreased in the first growing season and then continuously increased in the following planting years, reaching 8.26 g·kg−1 in the soils of 3 y, which was approximately 38.2% higher than that of 0 y. TN, TP, and SOM contents were high in soils around the drip line and decreased with distance from the drip line. In both horizontal and vertical directions, TN, TP, and SOM varied slightly in soils of 0, 1, and 2 y, while in soils of 3 y, TN and SOM decreased with increased distance in both horizontal and vertical directions and TP decreased obviously only within 10 cm in both directions. This indicated that the contents and distributions of soil nutrients in such saline-sodic soils could be improved with planting year under drip irrigation with local saline groundwater and especially around the drip line as the soil around the dripper was quickly ameliorated.
Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic ...sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.
Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has ...yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55-protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.
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CRISPR-mediated epigenome editing enables gene expression regulation without changing the underlying DNA sequence, and thus has vast potential for basic research and gene therapy. ...Effective selection of a single guide RNA (sgRNA) with high on-target efficiency and specificity would facilitate the application of epigenome editing tools. Here we performed an extensive analysis of CRISPR-mediated epigenome editing tools on thousands of experimentally examined on-target sites and established EpiCas-DL, a deep learning framework to optimize sgRNA design for gene silencing or activation. EpiCas-DL achieves high accuracy in sgRNA activity prediction for targeted gene silencing or activation and outperforms other available in silico methods. In addition, EpiCas-DL also identifies both epigenetic and sequence features that affect sgRNA efficacy in gene silencing and activation, facilitating the application of epigenome editing for research and therapy. EpiCas-DL is available at http://www.sunlab.fun:3838/EpiCas-DL.
The paddy field is a major source of gaseous carbon and nitrogen emissions, and reducing these emissions is of great significance for mitigating greenhouse effects and non-point source pollution in ...farmland. Biochar, derived from agricultural waste, possesses a stable structure, large specific surface area, abundant pore structures, and surface functional groups. These characteristics could enhance soil physicochemical properties and microbial activity, thereby facilitating the dual goals of increasing crop yield and reducing emissions. Based on numerous studies, this review summarizes the effects of biochar on the emissions of carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and ammonia volatilization (NH3), as well as on global warming potential (GWP) and greenhouse gas emission intensity (GHGI). It elucidates the mechanism of emission reduction by biochar amendment from the perspective of carbon and nitrogen conversion processes and soil physicochemical and biological properties. Numerous studies showed the application of 5~40 t ha−1 biochar reduced CO2, CH4, N2O, and NH3 emissions by 1.64~89.6%, 8.6~89.6%, 10~90%, and 12.27~53%, respectively. A small number of studies found that the application of 5~48 t ha−1 biochar increased CO2, CH4, N2O, and NH3 emissions by 12~37%, 19.85~176%, 13~84.23%, and 5.47~70.9%, respectively. Most scholars have found that biochar has varying degrees of emission reduction capabilities in different parts of the world. Therefore, future research directions have been suggested for utilizing biochar to reduce carbon and nitrogen emissions in paddy fields.
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