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Abstract Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.

Abstract Background Ductal carcinoma in situ (DCIS) of the breast is an early stage of breast cancer, and preventing its progression to invasive ductal carcinoma (IDC) is crucial for the early detection and treatment of breast cancer. Although single-cell transcriptome analysis technology has been widely used in breast cancer research, the biological mechanisms underlying the transition from DCIS to IDC remain poorly understood. Results We identified eight cell types through cell annotation, finding significant differences in T cell proportions between DCIS and IDC. Using this as a basis, we performed pseudotime analysis on T cell subpopulations, revealing that differentially expressed genes primarily regulate immune cell migration and modulation. By intersecting WGCNA results of T cells highly correlated with the subtypes and the differentially expressed genes, we identified six key genes: FGFBP2, GNLY, KLRD1, TYROBP, PRF1, and NKG7. Excluding PRF1, the other five genes were significantly associated with overall survival in breast cancer, highlighting their potential as prognostic biomarkers. Conclusions We identified immune cells that may play a role in the progression from DCIS to IDC and uncovered five key genes that can serve as prognostic markers for breast cancer. These findings provide insights into the mechanisms underlying the transition from DCIS to IDC, offering valuable perspectives for future research. Additionally, our results contribute to a better understanding of the biological processes involved in breast cancer progression.

Abstract Adenine base editors (ABEs) and cytosine base editors (CBEs) enable the single nucleotide editing of targeted DNA sites avoiding generation of double strand breaks, however, the genomic features that influence the outcomes of base editing in vivo still remain to be characterized. High-throughput datasets from lentiviral integrated libraries were used to investigate the sequence features affecting base editing outcomes, but the effects of endogenous factors beyond the DNA sequences are still largely unknown. Here the base editing outcomes of ABE and CBE were evaluated in mammalian cells for 5012 endogenous genomic sites and 11,868 genome-integrated target sequences, with 4654 genomic sites sharing the same target sequences. The comparative analyses revealed that the editing outcomes of ABE and CBE at endogenous sites were substantially different from those obtained using genome-integrated sequences. We found that the base editing efficiency at endogenous target sites of both ABE and CBE was influenced by endogenous factors, including epigenetic modifications and transcriptional activity. A deep-learning algorithm referred as BE_Endo, was developed based on the endogenous factors and sequence information from our genomic datasets, and it yielded unprecedented accuracy in predicting the base editing outcomes. These findings along with the developed computational algorithms may facilitate future application of BEs for scientific research and clinical gene therapy.

Abstract Bone metastasis is of common occurrence in renal cell carcinoma with poor prognosis, but no optimal treatment approach has been established for bone metastatic renal cell carcinoma. To explore the potential therapeutic targets for bone metastatic renal cell carcinoma, we profile single cell transcriptomes of 6 primary renal cell carcinoma and 9 bone metastatic renal cell carcinoma. We also include scRNA-seq data of early-stage renal cell carcinoma, late-stage renal cell carcinoma, normal kidneys and healthy bone marrow samples in the study to better understand the bone metastasis niche. The molecular properties and dynamic changes of major cell lineages in bone metastatic environment of renal cell carcinoma are characterized. Bone metastatic renal cell carcinoma is associated with multifaceted immune deficiency together with cancer-associated fibroblasts, specifically appearance of macrophages exhibiting malignant and pro-angiogenic features. We also reveal the dominance of immune inhibitory T cells in the bone metastatic renal cell carcinoma which can be partially restored by the treatment. Trajectory analysis showes that myeloid-derived suppressor cells are progenitors of macrophages in the bone metastatic renal cell carcinoma while monocytes are their progenitors in primary tumors and healthy bone marrows. Additionally, the infiltration of immune inhibitory CD47 + T cells is observed in bone metastatic tumors, which may be a result of reduced phagocytosis by SIRPA-expressing macrophages in the bone microenvironment. Together, our results provide a systematic view of various cell types in bone metastatic renal cell carcinoma and suggest avenues for therapeutic solutions.

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.

Abstract 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.

Abstract During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

Abstract N6-methyladenosine (m6A) modification has been shown to regulate RNA metabolism. Here, we investigate m6A dynamics during maternal-to-zygotic transition (MZT) in mice through multi-omic analysis. Our results show that m6A can be maternally inherited or de novo gained after fertilization. Interestingly, m6A 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 m6A readers for mouse preimplantation development. Our study reveals a key role of m6A 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.

Drip irrigation with saline water is frequently adopted to realize the sustainable utilization of saline–sodic soil with high water tables, and soil enzyme activities can be used to indicate changes in soil quality. In the current study, spatiotemporal changes in soil urease enzyme (URE), alkaline phosphatase (ALP) and invertase (INV) activities were investigated during consecutive growing seasons. Soil in beds was sampled before planting (0 y) and one, two, three and four years after the growing season (1 y, 2 y, 3 y, 4 y), and these samples were distributed 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 a distribution pattern of URE and ALP activities formed during the first growing season, while the distribution of INV activity formed until the third growing season. All three soil enzyme activities in the upper soil layers and positions close to the drip line were more greatly affected by planting year. The average URE activity of the soil profile decreased slightly during the first year and increased by about 220% and decreased by 20% after reclamation for two and three years, and finally, it increased to 4.9 μg NH4+·g−1·h−1 at the end of the fourth growing season. ALP activity remained stable during the first two years and rapidly increased in the following years; in particular, in the fourth year, it reached 32.7 μg ph(OH)·g−1·h−1. INV activity increased continually with the number of years after planting and reached 1009.0 μg glu·g−1·h−1 at the fourth season’s end. An analysis of variance indicated that URE, ALP and INV activities varied insignificantly among the time points of 0 y, 1 y, 2 y and 3 y (p < 0.05), while they were significantly higher for 4 y than for 0 y and 1 y. In addition, all three enzyme activities of the soil profile had an exponentially increasing trend with the number of years after planting. These results indicated the soil quality in saline–sodic soils could be improved with time under drip irrigation with local saline groundwater, especially around the drip line.

Understanding the distribution of water and nitrate nitrogen in the soil profile is crucial for the reasonable operation of fertigation, and it is also fundamental for controlling and regulating nitrate nitrogen in the root zone, thereby meeting a crop’s requirements. The application rates of fertilizer and water directly influence this distribution of water and nitrate nitrogen. However, the effects in Aeolian sandy soil, a type of developing soil bordering deserts, remain ambiguous. In this study, field experiments for different drip fertigation treatments in Aeolian sandy soil were conducted to investigate the soil water distribution, as well as that of nitrate nitrogen. A completely randomized experimental design was implemented, encompassing three levels of irrigation amount: low (W1), medium (W2), and high (W3), and three levels of nitrogen application rate: low (F1), medium (F2), high (F3). After the completion of each irrigation treatment, soil samples were extracted at 10–20 cm intervals. The soil water and nitrate nitrogen contents in the profiles of these samples were measured. The experimental results revealed that increasing the nitrogen application rate facilitated the retention of greater amounts of water and nitrate nitrogen in the soil profile. However, with an increase in the nitrogen application rate, both soil water and nitrate nitrogen exhibited a radial tendency to move away from the drip emitter. Some moved upward and accumulated in surface soil near a ridge furrow, while some moved downward and remained in a deeper area approximately 30 cm horizontally from the emitter at depths of 40–60 cm. The uniformity of the water distribution decreased with increasing nitrogen application under low water conditions, with a reversal of this trend observed in medium and high water treatments. The effect of nitrogen application level on the uniformity of the nitrate nitrogen distribution was not significant. There was no significant correlation between the average soil water content and nitrate nitrogen content along the horizontal direction, however, a positive correlation existed in the vertical direction. In the whole profile, increasing the nitrogen application enhanced the correlation under low water conditions, but under medium and high water conditions, this trend was the opposite. This implies that, to avoid nitrate nitrogen leaching or limiting in a specific area, a moderate nitrogen application level is advisable. Under low water conditions, nitrogen application showed a positive effect on the nitrate nitrogen content, and a higher application is recommended. In cases of substantial water irrigation or rainy years, the nitrogen application rate should be decreased.

The potential off-target effects of long-term expression of base editors in vivo are unclear. Here the authors report SAFETI, Systematic evaluation Approach For gene Editing tools by Transgenic mIce, to examine off-target effects of base editors over time in mice, and see abnormal side effects.

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.

Abstract Background Personalized therapy has been at the forefront of cancer care, making cancer treatment more effective. Since cancer patients respond individually to drug therapy, predicting the sensitivity of each patient to specific drugs is very helpful to apply therapeutic agents. Traditional methods focus on node (molecular) information but ignore relevant interactions among different nodes, which has very limited application in complex situations, such as cancer drug responses in real clinical practice. Results Treatment evaluation with Quantified Network (TreeQNet) is a webserver which could predict sensitivity to drugs for patients through the innovative use of proteomic and phosphoproteomic network from tumor tissues. Conclusion TreeQNet service: http://bioinfo.ustc.edu.cn/ . TreeQNet source code: https://github.com/Really00/treeqnet-web-front/ .

It is now widely known that C-X3-C motif ligand 1 (CX3CL1) plays an essential part in the process of regulating pro-inflammatory cells migration across a wide range of inflammatory disorders, including a number of malignancies. However, there has been no comprehensive study on the correlation between CX3CL1 and cancers on the basis of clinical features. In order to investigate the potential function of CX3CL1 in the clinical prognosis and immunotherapy, I evaluated the expression of CX3CL1 in numerous cancer types, methylation levels and genetic alterations. I found CX3CL1 was differentially expressed in numerous cancer types, which indicated CX3CL1 may plays a potential role in tumor progression. Furthermore, CX3CL1 was variably expressed in methylation levels and gene alterations in most cancers according to The Cancer Genome Atlas (TCGA). CX3CL1 was robustly associated with clinical characteristics and pathological stages, suggesting that it was related to the degree of tumor malignancy and the physical function of patients. As determined by the Kaplan-Meier method of estimating survival, high CX3CL1 expression was associated with either favorable or unfavorable outcomes depending on the different types of cancer. It suggests the correlation between CX3CL1 and tumor prognosis. Significant positive correlations of CX3CL1 expression with CD4+ T cells, M1 macrophage cells and activated mast cells have been established in the majority of TCGA malignancies. Which indicates CX3CL1 plays an important role in tumor immune microenvironment. Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that the chemokine signaling pathway may shed light on the pathway for CX3CL1 to exert function. In a conclusion, our study comprehensively summarizes the potential role of CX3CL1 in clinical prognosis and immunotherapy, suggesting that CX3CL1 may represent a promising pharmacological treatment target of tumors.

The surface relaxivity for tight cores is commonly determined by the methods of average pore radius (ARS) or surface-to-volume ratio (SVR). The ARS method is time-consuming, and permanent damage to core samples will occur due to the injection of mercury. In this study, a non-destructive method is proposed, instead of the ARS method, to calculate the surface relaxivity of tight core samples based on T2 cut-off value. Firstly, surface relaxivity is calculated using the new method (pseudo T2 cut-off, PTC). Secondly, the calculated surface relaxivity of ARS and SVR methods are compared. Thirdly, the T2 spectrum is converted into pore diameter distribution by choosing appropriate surface relaxivity. Finally, residual oil distribution can be obtained. The ultimate surface relaxivity values for tight sandstone core samples are 5.85, 2.98, 4.66, and 2.17 μm/s. Moreover, we obtained the pore diameter distribution in mesopores and macropores by jointly using these three methods. Residual oil is mainly distributed in micropores and mesopores. The new method proposed in this study is nondestructive and is helpful to quickly and efficiently determine the surface relaxivity of tight cores.

Herein a new approach to the application of agricultural eutrophic wastewater for rice plant cultivation is described. Biochar was used as a medium for the sorption of ammonia from simulated wastewater and subsequently as a nitrogen (N) releaser in the cultivation of rice plants. The main goals of this approach were to isolate ammonia from simulated wastewater and transfer it into rice cultivation, and or explore how exogenous N promoted the growth of rice. The results demonstrate that according to X-ray diffraction phase analysis, most of the properties of biochar were retained before and after loading NH+4-N. Compared with biochar, the crystal peak of AlOOH in N-loaded biochar (NLB) disappeared and the intensity of the crystal peak of CuCaSe2 decreased, which was the important mechanism allowing it to adsorb 30.8% of the N present in simulated low N-concentration agricultural wastewater. The soil N content in NLB treatments was higher than in Non-NLB treatments during the critical tillering and reproductive growth stages. Moreover, the N adsorption-desorption process of NLB matched the N requirements of the rice plant, and thus greatly increased the tiller number by 11.9% and rice yield by 7.5%. These results indicated that the indirect use of ammonia derived from wastewater using biochar as a sorption and releasing medium for rice plant cultivation was promising. This is the first time that biochar was used for possibly indirect reuse of agricultural eutrophic wastewater and enhancement of rice plant growth. [ABSTRACT FROM AUTHOR]