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CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

Background Glioma is the most common primary intracranial tumour and has a very poor prognosis. Pyroptosis, also known as inflammatory necrosis, is a type of programmed cell death that was discovered in recent years. The expression and role of pyroptosis-related genes in gliomas are still unclear. Methods In this study, we analysed the RNA-seq and clinical information of glioma patients from The Cancer Genome Atlas (TCGA) database and Chinese Glioma Genome Atlas (CGGA) database. To investigate the prognosis and immune microenvironment of pyroptosis-related genes in gliomas, we constructed a risk model based on the TCGA cohort. The patients in the CGGA cohort were used as the validation cohort. Results In this study, we identified 34 pyroptosis-related differentially expressed genes (DEGs) in glioma. By clustering these DEGs, all glioma cases can be divided into two clusters. Survival analysis showed that the overall survival time of Cluster 1 was significantly higher than that of Cluster 2. Using the TCGA cohort as the training set, a 10-gene risk model was constructed through univariate Cox regression analysis and LASSO Cox regression analysis. According to the risk score, gliomas were divided into high-risk and low-risk groups. Survival analysis showed that the low-risk group had a longer survival time than the high-risk group. The above results were verified in the CGGA validation cohort. To verify that the risk model was independent of other clinical features, the distribution and the Kaplan-Meier survival curves associated with risk scores were performed. Combined with the characteristics of the clinical cases, the risk score was found to be an independent factor predicting the overall survival of patients with glioma. The analysis of single sample Gene Set Enrichment Analysis (ssGSEA) showed that compared with the low-risk group, the high-risk group had immune cell and immune pathway activities that were significantly upregulated. Conclusion We established 10 pyroptosis-related gene markers that can be used as independent clinical predictors and provide a potential mechanism for the treatment of glioma.

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156 -mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox. Base editors are a powerful but underexplored tool for genetic engineering and directed evolution of plants. Here, the authors investigate the editing efficiency and specificity of TadA-8e-derived cytosine base editors and dual base editor TadDE in rice and tomato.

Background Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T 0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. Conclusions Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

Cold stratification is commonly used for breaking seed dormancy in plants. However, the effects of cold stratification on dormancy release and its physiological characteristics are still unclear in rice. Dormancy release by different periods (6, 12, 18 and 24 days) of cold stratification (4 °C) was conducted using japonica Jiucaiqing (Oryza sativa L) in this study. Physiological characteristics, including hormones, antioxidant enzyme and α-amylase activities, were further estimated in imbibed seeds after 12 and 24 days cold stratification. Seed dormancy of Jiucaiqing was completely released by cold stratification for 18 and 24 days. The expressions of the hormones, including abscisic acid (ABA), gibberellin (GA) and indole acetic acid (IAA), metabolism related genes OsCYP707A5, OsCYP707A7, OsGA20ox1, OsGA20ox2, OsGH3-2, OsGH3-7 and OsGH3-8 were significantly increased while OsNCED3 and OsZEP significantly decreased in imbibed seeds by cold stratification, which resulted in the rapid decline of ABA and the increases of GA3 and IAA. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), were significantly increased in imbibed seeds by cold stratification, which resulted in a slight increase of H2O2 level. The ratios of GA/ABA, GA/IAA and IAA/ABA and H2O2 level were gradually enhanced in imbibed seeds with cold stratification, which might contribute to the increase of α-amylase activity to promote dormancy release in rice.

Background: G-protein coupled receptor 34 (GPR34) is involved in cell motility, differentiation, and mitosis. GPR34 was reported to be highly expressed and play an oncogenic role in several solid tumors. Here, we investigated the mechanisms underlying how GPR34 promotes glioma progression. Methods: Bioinformatic analysis was performed on RNA-seq and clinical data from the gene expression omnibus (GEO), cancer genome atlas (TCGA), and Genotype-Tissue Expression (GTEx) databases. TIMER database and single-sample GSEA (ssGAEA) method were used to investigate the association between the GPR34 expression and immune infiltration level in glioma. Cox regression analysis was employed to ascertain whether the risk signature was an independent prognostic indicator for glioma. The viability and migratory/invasive potential of glioma cells were assessed using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Results: We found that GPR34 expression was positively correlated with immune infiltration level and that high GPR34 level may be associated with poor prognosis in glioma. We further found that GPR34 may serve as an independent prognostic marker and prediction factor for the clinicopathological features of glioma. We showed that knocking down GPR34 attenuated the viability and migratory/invasive capacity of glioma cells (U251 and LN229), while GPR34 overexpression exerted the opposite effects. Additionally, core enrichment in the GSEA analysis indicated that GPR34-mediated gliomagenesis was associated with the cell cycle arrest, epithelial–mesenchymal transition (EMT), and activation of the TGF-β/Smad pathway; furthermore, inhibiting TGF-β/Smad signaling using LY2157299, a TGF-β inhibitor, reversed the oncogenic effects and malignant phenotype associated with GPR34 overexpression. Conclusion: GPR34 enhances the malignancy and carcinogenesis of glioma by promoting an EMT-like process, G1/S phase cell cycle transition, and TGF-β/Smad signaling. Accordingly, GPR34 likely functions as an oncogene in glioma and may represent a potential therapeutic target for this cancer.

Background Glioma is the most common primary malignant tumor of the central nervous system, and due to the limited effectiveness of traditional single-target therapies, there is an urgent need for new therapeutic targets. Centromere protein F (CENPF) belongs to the centromere protein family and is mainly involved in the regulation of the cell cycle. CENPF has recently been found to play a key role in tumorigenesis and tumor progression, but its role in gliomas has not been well studied. Methods The expression level and clinical information of CENPF were obtained by analyzing the TCGA, CGGA and GEO databases. Immunohistochemistry and western blot analysis were used to quantitatively detect the expression of CENPF in glioma tissues and cell lines. Gene set enrichment analysis (GSEA) of TCGA and GSE16011 datasets was used to explore the molecular mechanism of the CENPF. CENPF-interacting proteins were detected by molecular docking and co-immunoprecipitation (Co-IP). After silencing CENPF, CCK-8 assay, Transwell assay and flow cytometry were used to detect changes in cell proliferation, invasion, cell cycle and apoptosis, and Western blot was used to detect changes in signaling pathway protein levels. Results Bioinformatics analysis showed that CENPF was generally highly expressed in gliomas and was associated with poor prognosis. This result was confirmed in glioma samples from our hospital. Multivariate Cox regression analysis showed that CENPF was an independent prognostic marker for gliomas. Western blot analysis in vitro showed that CENPF was overexpressed in the U251 and LN229 cell lines; therefore, these two cell lines were selected for subsequent experiments. GSEA analysis showed that CENPF was mainly involved in the G2/M phase-mediated cell cycle and P53 signaling pathway. Flow cytometry analysis confirmed that silencing CENPF induced G2/M phase arrest and increased apoptosis in glioma cells. Subsequent experiments confirmed that CENPF influences the epithelial-mesenchymal transition (EMT) process through the mTORC1 signaling pathway. Molecular docking and Co-IP assay revealed that CENPF exerts its effects by interacting with PLA2G4A promoting the downstream signaling pathway. Finally, we found that silencing CENPF combined with a PLA2G4A inhibitor (AACOCF3) induced glioma cell apoptosis and exhibited anti-glioma effects. Conclusions This study found that CENPF plays a key role in promoting tumorigenesis through its interaction with PLA2G4A. This study provides a theoretical foundation for advancing multi-targeted therapies in glioma and for developing strategies to overcome tumor drug resistance.

As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type‐V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1‐RRA variant did not perform better than the wild‐type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants. Core Ideas Exploration of a novel compact clustered regularly interspaced short palindromic repeats‐Cas system in plants. Testing of uncultured archaeon Cas12f1 in both monocot and dicot plants. Improved genome editing with polymerase III promoters for guide RNA expression. Plain Language Summary Plant genome editing is a promising breeding technology. Many clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas systems have been discovered and engineered as tools for genome editing. Most of the existing CRISPR‐Cas nucleases and genome editing systems are based on Cas9 and Cas12a, which are large proteins. The Hypercompact CRISPR‐Cas system could enable more innovative delivery approaches. Here, we explored uncultured archaeon Cas12f1 (Un1Cas12f1), a type V‐F miniature CRISPR system, for genome editing in plants. We developed an efficient CRISPR‐Un1Cas12f1 expression system that is based on an optimized guide RNA scaffold and polymerase III small RNA promoters. We further characterized Un1Cas12f1's sequence preference and editing outcomes in rice and tomato protoplasts by next‐generation sequencing. This is a comprehensive study of the Un1Cas12f1 system in plants. Further improvement of Un1Cas12f1 is warranted before it becomes a powerful tool for genome editing in plants.