Preparation of a miR-155-activating nucleic acid nanoflower to study the molecular mechanism of miR-155 in inflammation.

At present, the molecular mechanisms underlying inflammation remain unclear. In recent years, research on inflammation has focused on stimulating cell inflammation by using exogenous pro-inflammatory substances such as lipopolysaccharide (LPS) or inflammatory factors. To investigate the molecular mechanism of inflammation from a new perspective, we designed a nucleic acid nanoflowers (NFs) complex to directly activate inflammatory genes to study the inflammatory response without the need for external microbial factors to trigger an inflammatory response. An RNAa-type target gene-activated NFs was designed. Human umbilical vein endothelial cells (HUVECs) were transfected with NFs carrying small activating RNA (saRNAs) to directly co-activate microRNA (miR)-155 and SHIP1 genes. After RNA activation (RNAa)-type NFs were transferred into HUVECs, the expression of miR-155 and pro-inflammatory and cancer-related factors increased, anti-inflammatory factors were reduced, cell proliferation increased, and cell migration was promoted. IL-1ß protein levels were decreased and SHIP1 expression was downregulated. When miR-155 and its target SHIP1 were both activated, the expression of both was unaltered, maintaining cell homeostasis. This points towards miR-155 overexpression can trigger inflammation, and that miR-155 and its target genes act as a molecular switch role in the development of inflammation.

Synthesis of quinazoin-4-ones through an acid ion exchange resin mediated cascade reaction.

An interesting cascade reaction of N-(2-(4,5-dihydrooxazol-2-yl)phenyl)benzamide in the presence of an acid ion exchange resin is described. In this reaction, a range of substrates bearing various substituent groups are well compatible. This work provides a green and atom-economical alternative approach for the synthesis of quinazolin-4-ones in good yields.

Inhibition of integrin ß3, a binding partner of kallistatin, leads to reduced viability, invasion and proliferation in NCI-H446 cells.

BACKGROUND: Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear. METHODS: Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin ß3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used. RESULTS: Interestingly, kallistatin was capable of binding integrin ß3 on the cell surface of NCI-H446 cells. Meanwhile, integrin ß3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin ß3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation. CONCLUSIONS: These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin ß3, leading to blockade of the related signaling pathway.

[Anti-inflammatory effect of recombinant human kallistatin in ulcerative colitis of mice].

This study was designed to evaluate the anti-inflammatory effect of recombinant human kallistatin (Kal) on ulcerative colitis (UC) in the mouse model. Acute colitis was induced by administration of 4% dextran sodium suffate (DSS) to KM mice for 7 days. The mice were then randomized into 5 groups: model control, Kal 0.2 mg·kg(-1)·d(-1), 1.0 mg·kg(-1)·d(-1) and 2.0 mg·kg-1·d(-1) group, salazosulfapyridine (SASP) group. Ten age-matched normal KM mouse were administered with saline in the normal control. The weight, colon length, inflammation factor (MPO/SOD/MDA) and TNF-α/IL-10 levels among the five groups of mice were determined. The results showed that histological index score and MPO/MDA/TNF-α levels of high-dose Kal treatment group and SASP group were significantly lower compared with the model group (P < 0.01), but the weight, colon length, IL-10 level and SOD activity were significant higher than the model group (P < 0.01), approaching the normal group. These parameters showed that Kal can significantly relieve the UC state in a dose-dependent manner. This study demonstrates that Kal significantly remits UC in mice, and participates in the regulation of inflammatory cytokines TNF-α/IL-10 levels and has some antioxidant activity.

Creation of DNA aptamers against recombinant bone morphogenetic protein 15.

The oocyte-derived growth factor bone morphogenetic protein (BMP) 15 plays important roles in fertility, but its mechanism of action differs between species. Generation of BMP15-binding molecules, as an essential investigation tool, would be helpful to provide valuable insight into the underlying biological features of BMP15. The BMP15-binding molecules could be antibodies or aptamers. Aptamers have many advantages over antibodies as macromolecular ligands for target proteins. DNA aptamers can be obtained by a method of Systematic Evolution of Ligands by EXponential enrichment (SELEX) beginning with a pool of random sequences. However, the success of this technique cannot be guaranteed if the initial pool lacks candidate sequences. Herein, we report on the creation of DNA aptamers by means of modified SELEX. The modification included enhanced mutation and progressive selection during an in vitro evolutionary process. As a proof-of-principle, we started from a single sequence instead of a multiple-sequence pool. Functional aptamers against the recombinant BMP15 were successfully created and identified.

Associations between the use of red yeast rice preparations and adverse health outcomes: An umbrella review of meta-analyses of randomized controlled trials.

BACKGROUND: Red yeast rice (RYR), a natural lipid-lowering agent, is widely used in clinical practice. However, the existing meta-analyses concerning the safety of RYR preparations have yielded inconsistent results, and the credibility of the evidence has not been quantified. OBJECTIVE: This study was designed to evaluate the existing evidence and offer a comprehensive understanding of the associations between the use of RYR preparations and various adverse health outcomes. SEARCH STRATEGY: Seven literature databases were searched from inception to May 5, 2023, using medical subject headings and free-text terms (e.g., "red yeast rice," "Xuezhikang," and "Zhibitai"). INCLUSION CRITERIA: Meta-analyses that investigated and quantitatively estimated associations between the use of RYR preparations and adverse health outcomes were included in this study. DATA EXTRACTION AND ANALYSIS: Two researchers independently extracted data using a standardized data collection table; any disagreements were resolved by consulting a third researcher. Based on the participant, intervention, comparator and outcome (PICO) framework in each eligible meta-analysis, a series of unique associations between the use of RYR preparations and adverse health outcomes were determined. The associations' effect estimates were re-evaluated using random-effect models. RESULTS: Fifteen meta-analyses, comprising 186 (164 unique) randomized controlled trials, were identified. Based on A MeaSurement Tool to Assess Systematic Reviews version 2, 3 (20%) and 12 (80%) of these meta-analyses had low and critically low confidence, respectively. A total of 61 unique associations between the use of RYR preparations and adverse health outcomes were extracted from eligible meta-analyses. Based on the random-effect models, 10 (16.4%) associations indicated a significant protective effect of RYR preparations against adverse health outcomes, while 5 (8.2%) indicated an increased risk of adverse health outcomes related to uric acid, alanine transaminase and aspartate transaminase levels. The other 46 (75.4%) associations showed no significant difference between the use of RYR preparations and control treatments. Regarding the credibility of the evidence, 21 (34.4%), 34 (55.7%) and 6 (9.8%) associations showed moderate, low and very low credibility, respectively. CONCLUSION: The evidence examined in this study suggests that RYR preparations are safe; however, the credibility of the evidence was not high. Further high-quality evidence is required. Please cite this article as: Ma ZY, Yang SP, Li Y, Xu TT, Yang YL, Yang HY, Li HB, Zhou LJ, Diao Y, Li SY. Associations between the use of red yeast rice preparations and adverse health outcomes: An umbrella review of meta-analyses of randomized controlled trials. J Integr Med. 2024; 22(2): 126-136.

Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing.

Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 µL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).

[Recent progress of the aptamer-based antiviral drugs].

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.

Development and validation of prediction models for the prognosis of colon cancer with lung metastases: a population-based cohort study.

Background: Current studies on the establishment of prognostic models for colon cancer with lung metastasis (CCLM) were lacking. This study aimed to construct and validate prediction models of overall survival (OS) and cancer-specific survival (CSS) probability in CCLM patients. Method: Data on 1,284 patients with CCLM were collected from the Surveillance, Epidemiology, and End Results (SEER) database. Patients were randomly assigned with 7:3 (stratified by survival time) to a development set and a validation set on the basis of computer-calculated random numbers. After screening the predictors by the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression, the suitable predictors were entered into Cox proportional hazard models to build prediction models. Calibration curves, concordance index (C-index), time-dependent receiver operating characteristic (ROC) curves, and decision curve analysis (DCA) were used to perform the validation of models. Based on model-predicted risk scores, patients were divided into low-risk and high-risk groups. The Kaplan-Meier (K-M) plots and log-rank test were applied to perform survival analysis between the two groups. Results: Building upon the LASSO and multivariate Cox regression, six variables were significantly associated with OS and CSS (i.e., tumor grade, AJCC T stage, AJCC N stage, chemotherapy, CEA, liver metastasis). In development, validation, and expanded testing sets, AUCs and C-indexes of the OS and CSS prediction models were all greater than or near 0.7, which indicated excellent predictability of models. On the whole, the calibration curves coincided with the diagonal in two models. DCA indicated that the models had higher clinical benefit than any single risk factor. Survival analysis results showed that the prognosis was worse in the high-risk group than in the low-risk group, which suggested that the models had significant discrimination for patients with different prognoses. Conclusion: After verification, our prediction models of CCLM are reliable and can predict the OS and CSS of CCLM patients in the next 1, 3, and 5 years, providing valuable guidance for clinical prognosis estimation and individualized administration of patients with CCLM.

Chemical kinetic investigation on NOx emission of SI engine fueled with gasoline-ethanol fuel blends.

Understanding the nitrogen oxide (NOx) formation chemical kinetic mechanism and analyzing the effect of relevant influence parameters are the effective strategy for NOx emission control. Based on the essential role of ethanol-gasoline blends among oxygenate alternative fuel, the experiments in a GDI (gasoline direct injection) SI (spark ignition) engine and the chemical kinetic simulation were carried out. According to the validated model, seven NO contributing reactions and three reaction pathways were observed. Besides the thermal NO formation pathway, two other pathways with N2O and NNH through NH-HNO-NO have nonnegligible places in the high engine speed condition. As for the parameters, initial temperature aggravates NO emission, initial pressure and ethanol fraction inhibit NO, which influence it through thermal NO pathway and have slight impact on the other two pathways. While with the increase of equivalence ratio (ER) from 0.5 to 1.0, ER promotes first and then resists NO formation, getting highest emission when ER equals to 0.85. In a lean burn condition, the thermal pathway is highly inhibited and the N2O pathway is sharply accelerated. Through current work, NOx producing mechanism under high-speed condition is investigated comprehensively, which not only completes the total NO formation pathways from atmospheric N2 but also provides reference for the designing and modification of low harmful NOx emitting gasoline-ethanol engines.

Expression and Functional Study of BcWRKY1 in Baphicacanthus cusia (Nees) Bremek.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an important medicinal plant. Its effective substances including indigo and indirubin are metabolites in indoleacetate metabolic pathway. Based on a previous transcriptome sequencing analysis, a WRKY transcription factor, BcWRKY1, in B. cusia was identified, showing significant correlation with effective substances from B. cusia. In this study, BcWRKY1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the BcWRKY1 gene was 916 bp in length, containing three exons and two introns. The open reading frame (ORF) of BcWRKY1 was 534 bp in length and encoded a WRKY domain-containing protein with 177 amino acids residues. Subcellular localization showed that BcWRKY1 protein was mainly localized in the nucleus. It could bind to the W-box motif and its role in transcriptional activation was confirmed in yeast. The function of BcWRKY1 was investigated by overexpressing BcWRKY1 in Arabidopsis thaliana. Metabolic profiles in wild type and BcWRKY1-OX1 transgenic Arabidopsis thaliana were analyzed with LC-MS. Results showed that the metabolic profile was significantly changed in BcWRKY1-OX1 transgenic Arabidopsis thaliana compared with wild type. Furthermore, indole-related metabolites were significantly increased in BcWRKY1-OX1 transgenic Arabidopsis thaliana, and the metabolic pathway analysis showed that flavonoid biosynthesis was significantly enriched. Overexpression of BcWRKY1 significantly changed flavonoid and indole metabolism and indole-related metabolites were significantly upregulated. We postulated that the BcWRKY1 transcription factor might be involved in the regulation of effective substances metabolism in B. cusia.

Resistive Switching Memristor: On the Direct Observation of Physical Nature of Parameter Variability.

Ion-based memristive switching has attracted widespread attention from industries owing to its outstanding advantages in storage and neuromorphic computing. Major issues for achieving brain-inspired computation of highly functional memory in redox-based ion devices are relatively large variability in their operating parameters and limited cycling endurance. In some devices, volatile and nonvolatile operations often replace each other without changing operating conditions. To address these issues, it is important to observe directly what is happening in repeated operations. Herein, we use a planar device that enables direct capturing of microscopic behaviors in the nucleation and growth of metal whiskers under repeated switching to verify the microscopic origin of the large parameter variability. We report direct observations that reveal the physical origin for the large cycle-to-cycle and device-to-device variability in memristive switching, which was achieved using planar polymer atomic switches with a gap >1 µm. We find that the deposition location of metal atoms is closely related to the crystallinity of the ion transport layer (solid polymer electrolyte, SPE). The filament variability (shape, position, quantity, etc.) during different cycles and devices is indeed the main reason for the observed variability in the operating characteristics. The results shed unique light on the complexity of the operation of the ion device, that is, the evolution of the dielectric layer and metal filament must be considered.

Synthesis of (E)-Quinoxalinone Oximes through a Multicomponent Reaction under Mild Conditions.

Herein, a novel method for the gram-scale synthesis of (E)-quinoxalinone oximes through a multicomponent reaction under mild conditions is described. Such a transformation was performed under transition-metal-free conditions, affording (E)-oximes in a moderate-to-good yield through recrystallization. Our methodology demonstrates a successful combination of a Mannich-type reaction and radical coupling, providing a green and practical approach for the synthesis of potentially bioactive quinoxalinone-containing molecules.

Effects of Selenium-Enriched Yeast on Performance, Egg Quality, Antioxidant Balance, and Egg Selenium Content in Laying Ducks.

This study investigated the effects of dietary selenium-enriched yeast (Se yeast) supplementation on the laying performance, egg quality, plasma antioxidant balance, and egg selenium (Se) content in laying Longyan ducks. A total of 480 32-week-old ducks were randomly divided into four dietary treatments, each consisting of six replicates of 20 ducks. The dietary treatments were a control basal diet and basal diets with supplementation of 0.05, 0.15, and 0.25 mg Se/kg via Se yeast. The analyzed Se contents of the four diets were 0.15, 0.21, 0.36, and 0.43 mg Se/kg, respectively. Dietary Se yeast supplementation had no apparent effects on laying performance and egg quality (p > 0.05), but it improved the antioxidant balance of ducks, as inferred by greater glutathione peroxidase and catalase activities, and decreased the malondialdehyde content in plasma of ducks (p < 0.05). It was suggested that the Se content in the basal diet containing 0.15 mg/kg of Se requirement is adequate for productive performance, but not for the antioxidant balance of laying ducks. Besides that, the Se contents in the yolk, albumen, and whole egg increased linearly as the Se supplementation levels increased. With more feeding days, the Se contents in the yolk and whole egg from non-Se-yeast-supplemented ducks increased linearly (p < 0.05), while those from Se-yeast-supplemented ducks showed a quadratic relationship (p < 0.05). In conclusion, the Se content of the basal diet at 0.15 mg/kg was adequate for laying performance and egg quality traits in laying ducks. Dietary Se yeast supplementation is beneficial to improve the antioxidant balance of laying ducks and increase the Se deposition in eggs for producing Se-enriched eggs. Based on the quadratic model or the quadratic broken-line model analyses, supplemental 0.19 mg Se/kg via Se yeast, with a total equivalent of 0.34 mg Se/kg in the diet, could provide the optimum antioxidant balance in laying ducks. Dietary supplementation of 0.25 mg Se/kg via Se yeast, with a total equivalent of 0.40 mg Se/kg in the diet, could lead to achieving the desired Se content in the whole egg.

Controlled Growth of Fine Multifilaments in Polymer-Based Memristive Devices Via the Conduction Control.

Solid polymer electrolyte (SPE) is one of the choices for many ionic devices, including organic transistors, ion batteries, memristors, and more. However, uncontrollable conductive filament formation seriously affects the performance of the device. In this paper, the PEDOT:PSS was doped to improve the electronic and ionic conductivity of amorphous polymer electrolyte poly(vinylpyrrolidone) (PVP), realizing the transition of the filaments growth from cathode to anode in atomic switch memristors. Based on the difference in ion and electron mobility and scanning electron microscope observation, the in situ reductions of metal ions inside the dielectric layer can effectively prevent the formation of uncontrollable filaments. The formation of uniformly distributed metal particles in the dielectric layer is similar to co-sputter doping technology, which enables the device to exhibit excellent performance, such as smaller set/reset bias distribution, endurance, and retention. Obviously, this innovative way improves the conductive mechanism of ionic devices.

SiO2/Ta2O5 heterojunction ECM memristors: physical nature of their low voltage operation with high stability and uniformity.

Non-uniformity of switching parameters, e.g., switching voltage and resistances of the high resistance state and low resistance state, could obstruct the practical application of electrochemical metallization cells. Higher switching voltages are also undesirable in terms of power consumption. The non-uniformity is usually related to the number of conductive filaments (CFs) and their degree of dissolution during the RESET process. The number of CFs can be ideally reduced to one by decreasing the device area. However, the degree and location of the dissolution of CFs are difficult to control. Here, we introduce a SiO2/Ta2O5 heterojunction to control the dissolution of CFs, in which the growth direction and the shape of CFs are controlled by the SiO2 layer, while the dissolution of CFs is controlled in the ultrathin Ta2O5 layer. Transmission electron microscopy analysis clearly suggested that the formation/dissolution of CFs occurs in the ultrathin Ta2O5 layer, resulting in low voltage operation (<0.3 V) with high stability and uniformity (Vset distributes in the range smaller than 0.1 V and Vreset distributes in the range smaller than 0.08 V).

MiR-25-3p Serves as an Oncogenic MicroRNA by Downregulating the Expression of Merlin in Osteosarcoma.

PURPOSE: Moesin-ezrin-radixin-like protein (Merlin) has been identified as a tumor suppressor in several types of cancers. However, the biological function of Merlin in osteosarcoma remains unclear. MicroRNAs (miRNAs) can influence cancer progression by targeting oncogenes or anti-oncogenes. In this study, we sought to evaluate the regulation of Merlin expression by miR-25-3p and the role of the miR-25-3p/Merlin axis in osteosarcoma progression, with the aim of identifying a potential therapeutic target for osteosarcoma. MATERIALS AND METHODS: TCGA (The Cancer Genome Atlas) database was used to analyze the correlation between Merlin expression and prognosis. RT-qPCR and Western blotting analyses were performed to compare Merlin expression between normal and malignant cells. A dual-luciferase reporter assay was performed to evaluate the direct targeting of Merlin by miR-25-3p. We overexpressed miR-25-3p, or/and Merlin, in U-2 OS and 143B cells, and studied their cellular functions in vitro. MTT and colony formation assays were performed to determine the effects on cell growth. EdU and cell cycle assays were performed to analyze the effects in cell replication. We used annexin V-fluorescein isothiocyanate and propidium iodide to stain apoptotic cells, and analyzed the cells using flow cytometry. The effects on cell metastasis were studied in wound healing and transwell assays. Lastly, the underlying mechanism was determined in RT-qPCR and Western blotting experiments. RESULTS: Low Merlin expression was linked to poor prognosis. miR-25-3p was observed to directly target Merlin and downregulate its expression. miR-25-3p promoted cell growth, migration, and invasion, and inhibited apoptosis induced by cisplatin. Moreover, the overexpression of Merlin reversed the abovementioned effects of miR-25-3p. Further, the miR-25-3p/Merlin axis was observed to play an important role in the Hippo pathway, and regulated the expression of genes such as BIRC5, CTGF, and CYR61. CONCLUSION: miR-25-3p functions as an oncogenic microRNA in osteosarcoma by targeting Merlin, and may serve as a potential therapeutic target for osteosarcoma.

Overexpression of OsFTL10 induces early flowering and improves drought tolerance in Oryza sativa L.

Flowering time control is critically important for the reproductive accomplishment of higher plants as floral transition can be affected by both environmental and endogenous signals. Flowering Locus T-like (FTL) genes are major genetic determinants of flowering in plants. In rice, 13 OsFTL genes have been annotated in the genome and amongst them, Hd3a (OsFTL2) and RFT1 (OsFTL3) have been studied extensively and their functions are confirmed as central florigens that control rice flowering under short day and long day environment, respectively. In this report, a rice OsFTL gene, OsFTL10, was characterized, and its function on flowering and abiotic stress was investigated. The expression level of OsFTL10 was high in young seedlings and shown to be induced by GA3 and drought stress. Overexpression of OsFTL10 resulted in earlier flowering in rice plants by up to 2 weeks, through up-regulation of the downstream gene OsMADS15. OsFTL10 also regulated Ehd1 and OsMADS51 through a feedback mechanism. The OsFTL10 protein was also detected in both nucleus and cytoplasm. Furthermore, yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) results show that OsFTL10 could interact with multiple 14-3-3s, suggesting that OsFTL10 might function in a similar way to Hd3a in promoting rice flowering by forming a FAC complex with 14-3-3, and OsFD1. Further experiments revealed that constitutive expression of OsFTL10 improved the drought tolerance of transgenic plants by stimulating the expression of drought responsive genes. These results suggest that rice FTL genes might function in flowering promotion and responses to environmental signals.

Transition-Metal and Solvent-Free Oxidative C-H Fluoroalkoxylation of Quinoxalinones with Fluoroalkyl Alcohols.

The first example of oxidative C-H fluoroalkoxylation of quinoxalinones with fluoroalkyl alcohols under transition-metal and solvent-free conditions is described. This approach provides the synthesis of fluoroalkoxylated quinoxaline derivatives with good to excellent yields under mild reactions conditions. This method can also be extended to the facile and efficient synthesis of histamine-4 receptor.

[Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELâ crude extracts].

CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.