COLLOIDS AND SURFACES B-BIOINTERFACES
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2023
·
IF 5.8
Wenbo Wang,Shihao Zhou,Zhaoyi Cheng,Dong Ma,Tao Liu
CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin α v β 3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.
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BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Jienan Shen,Zhi Chen,Ruibin Xie,Jingfeng Li,Chunyan Liu,Yaqing He,Xiaopeng Ma,Hui Yang,Zhongjian Xie
Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H 1 N 1 , H 3 N 2 , IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.
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ACS Sensors
·
2022
·
IF 9.618
Jiahao Li,Lina Tang,Tingxian Li,Kun Li,Yulin Zhang,Wei Ni,Meng-Meng Xiao,Youyun Zhao,Zhi-Yong Zhang,Guo-Jun Zhang
The path toward field-effect transistor (FET) application from laboratory to clinic has delivered a compelling push in the biomedical domain, yet ultrasensitive and timely pathogen identification without PCR remains a long-lasting challenge. Herein, we create a generic check station termed “CRISPR-FET”, first incorporating the CRISPR/Cas13a system within the FET modality, for accelerated and unamplified detection of viral RNA. Unlike conventional FETs bearing target-specific receptors, this sensor holds three unique advancements: (i) an ingenious sensing mechanism is used, which converts the signal of a large-sized analyte into an on-chip cleavage response of an immobilized CRISPR reporter, enabling signal generation events to occur all within the Debye length; (ii) the multipurpose inspection of the CoV ORF1ab, CoV N gene, and HCV RNA unveils the potential for “one-for-all” scalable FET-based molecular diagnostics; and (iii) it is shown that Cas13a-crRNAs targeting different sites of the viral genome can be deployed in tandem to amplify the FET response, empowering the detection limit down to 1.56 aM, which is a world-record level of sensitivity in the FET for direct viral gene sensing. Notably, a brilliant clinical applicability was made in distinguishing HCV-infected patients from normal controls. Overall, this study sheds new insights into FET-based nucleic acid sensing technology and invokes a vision for its possible future roles in diagnosis of various viral diseases.
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Pharmaceutics
·
2022
·Vol. 14
·期 11
·2307
·
IF 6.525
Xueyan Gou,Fenglin Ran,Jinru Yang,Yanrong Ma,Xin’an Wu
Background: Organic anion transporter 1 (OAT1) and OAT3 have an overlapping spectrum of substrates such that one can exert a compensatory effect when the other is dysfunctional. As a result, the knockout of either OAT1 or OAT3 is not reflected in a change in the excretion of organic anionic substrates. To date, only the mOAT1 and mOAT3 individual knockout mouse models have been available. Methods: In this study, we successfully generated aSlc22a6/Slc22a8double-knockout (KO) rat model using CRISPR/Cas9 technology and evaluated its biological properties. Results: The double-knockout rat model did not expressionmRNAfor rOAT1 or rOAT3 in the kidneys. Consistently, the renal excretion ofp-aminohippuric acid (PAH), the classical substrate of OAT1/OAT3, was substantially decreased in theSlc22a6/Slc22a8double-knockout rats. The relativemRNAlevel ofSlco4c1was up-regulated in KO rats. No renal pathological phenotype was evident. The renal elimination of the organic anionic drug furosemide was nearly abolished in theSlc22a6/Slc22a8knockout rats, but elimination of the organic cationic drug metformin was hardly affected. Conclusions: These results demonstrate that this rat model is a useful tool for investigating the functions of OAT1/OAT3 in metabolic diseases, drug metabolism and pharmacokinetics, and OATs-mediated drug interactions.Keywords:OAT1/OAT3;CRISPR/Cas9;gene knockout;pharmacokinetic
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ANALYTICA CHIMICA ACTA
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2022
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IF 6.911
Mengyang Zhou,Haoyu Wang,Chao Li,Chao Yan,Panzhu Qin,Lin Huang
Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.
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FOOD CHEMISTRY
·
2023
·
IF 8.8
Xuening Shi,Hang Li,Shuo Yao,Yukun Ding,Xiuzhu Lin,Hui Xu,Yi Liu,Chao Zhao,Tong Zhang,Juan Wang
The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans -cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.
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STAR Protocols
·
2022
·
IF 0
Meixia Wang,Fang Yan,Huanbin Zhou
Summary Base editing is a precision genome-editing approach that is widely utilized to generate single-nucleotide variants (SNVs) in genomes. Here, we present a protocol to perform targeted adenine (A)-to-guanine (G) substitution in rice using adenine base editor (ABE). We detail the design of sgRNA, CRISPR plasmid construction, rapid genetic transformation of rice, and genotyping of editing events. This protocol can be applied to cytosine base editing in rice as well. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021). 1
研究领域:
植物生物学
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基因组编辑
·
分子遗传学
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IMMUNITY
·
2021
·
IF 31.745
Run Fang,Qifei Jiang,Yukun Guan,Pengfei Gao,Rui Zhang,Zhen Zhao,Zhengfan Jiang
Summary Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O -linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.
研究领域:
免疫学
·
细胞生物学
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分子生物学
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Yining Zhao,Ziye Liu,Ziting Liu,Juan Zhang,Jinghui Peng,Genxi Li
Prosthetic joint infection (PJI), which is still difficult to be diagnosing, is caused by bacterial infection. Rapid detection for trace amounts of bacteria should be a solution; however, the existing assays cannot serve for PJI diagnosis due to the inherent limitations. Herein, a strategy based on supramolecular chemistry mediated Cas12a catalytic aggregation (CCA) is explored in this work for ultrasensitive detection of universal bacteria. In the design, supramolecular chemistry is introduced to impede the cis-cleavage of activated Cas12a, resulting in its aggregation in the surrounding of bacteria with the linkage of CCA probe. Since Cas12a aggregation owns excellently enhanced catalytic efficiency, the CCA-based strategy can be used for bacterial analysis with a limit of detection as low as 1 CFU/mL. With high sensitivity, reproducibility, generalizability, and stability, the CCA-based strategy can be applied for the detection of clinical effusion samples. The remarkable performance of CCA-based strategy has also demonstrated its robust potential to be a simple, rapid, and highly sensitive bacteria diagnostic tool, paving the way for prompt diagnosis and management of PJI.
研究领域:
微生物学
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分子诊断学
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生物医学工程
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转化医学
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感染性疾病
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纳米生物技术
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临床检验医学
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PLANT BIOTECHNOLOGY JOURNAL
·
2019
·Vol. 18
·期 7
·1550-1561
·
IF 6.84
Qi Gao,Huifeng Luo,Yongping Li,Zhongchi Liu,Chunying Kang
Summary Fruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles ( RAP ) gene encoding a glutathione S-transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca . The parent of rap was the F. vesca variety ‘Yellow Wonder’ that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa . Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up-regulated in RAP-ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10 . Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white-fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.
研究领域:
植物生物学
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遗传学
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分子生物学
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农学
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Cell Reports
·
2026
·Vol. 45
·期 8
·
IF 7.7
Jianhui Wang,Meiqi Liu,Yiming Chen,Jiancheng Chen,Xin Zhong,Zilong Wang,Xian Jiang,Ruilin Tian
While advances in omics profiling rapidly expand the catalog of genes associated with brain activity in health and disease, functional annotation lags far behind. Here, we establish a high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons. By converting cumulative neuronal activity into a stable, flow cytometry-readable signal, this approach enables systematic interrogation through pooled screening. Using a focused library of memory-associated genes, we recover known regulators and identify TMEM50A, a previously uncharacterized protein, as an essential regulator of neuronal activity. TMEM50A forms a complex with LEPROTL1 and associates with ESCRT-III machinery on multivesicular bodies (MVBs). TMEM50A loss impairs MVBs function, remodels the neuronal surface proteome, reduces synapse density, and alters behavior in mice. This platform enables systematic discovery of neuronal activity regulators and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.
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Advanced Science
·
2026
·
IF 14.1
Zi-Jian Huang,Feng-Ming Li,Yi-Fan Tu,Ke-Ke Feng,Cheng-Lei Li,Shi-Cheng Tian,Yong-Shan Hu,Jing-Wei Shao,Zhen-Hua Liu
Photothermal therapy (PTT), a non-invasive tumor treatment, shows promise but is limited in solid tumors by restricted tissue penetration, thermotolerance, anti-apoptotic and immunosuppressive effects. In this study, tumor microenvironment-responsive nanoplatform VARH was constructed based on MXene. Under NIR-II laser irradiation, VARH achieves a high photothermal conversion efficiency of 44.21%. Loaded 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride decomposes at high temperatures to generate alkyl radicals, synergizing with hydroxyl radicals from V 4+ -catalyzed endogenous H 2 O 2 decomposition, enabling chemodynamic therapy (CDT) and thermal dynamic therapy to enhance tumor cell oxidative damage. Triggered by high glutathione, VARH releases ribonucleoprotein (RNP) complexes to knockout heat shock protein 90 (HSP90), attenuating cellular heat resistance and promoting apoptosis. It also enhances T cell-mediated anti-tumor immunity and, with free radicals, promotes tumor cell immunogenic cell death (ICD), achieving immunotherapeutic multi-effect synergy. Integrating nanotechnology with precise gene editing, this study develops a novel multimodal synergistic therapy system, providing new insights for multi-modal treatment R&D and advancing PTT and free radical-based cancer therapies.
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