Current Protocols
·
2024
·Vol. 4
·期 4
·e1033
·
IF 0
Ruifeng Wang,Shuhua Ma,Nengwei Xu,Yumiao Gan,Pengya Li,Jingying Zhang,Zhixiang Zhang,Qingyang Gu,Jian Xiang
Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2 : Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol : Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3 : Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo
研究领域:
肿瘤学
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分子生物学
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基因编辑
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药理学
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DOI
ANALYTICA CHIMICA ACTA
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2025
·
IF 6
Zhaleh Asadi Fakhr,Wei Xie,Su Zeng,Sheng Cai
Background CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis–Menten kinetic parameters. Results Three ciRS-7–specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = –862, –813, and –615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA–target pair. Michaelis–Menten analysis showed k cat values of 1.39, 1.31, and 1.16 s -1 for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity. Significance Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.
研究领域:
分子诊断
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CRISPR技术
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RNA生物学
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生物化学工程
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即时检测
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Yuqian Guo,Yuanqian Xiong,Weipeng Tong,Xiaolin Huang,Yonghua Xiong
Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.
研究领域:
分子诊断
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食品安全监测
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纳米生物技术
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CRISPR技术
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生物传感
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产品关联
DOI
PubMed
高影响因子
ACS Omega
·
2025
·
IF 4.3
Na Du,Guochao Xu,Yao Zhang,Jing Hou,Ying Wang,Yiran Li,Bing Xu
Nosocomial infection is a major global cause of mortality, posing significant public health challenges. Recent studies have identified RNA biomarkers, including microRNA (miRNA) and circular RNA (circRNA), as promising diagnostic tools for nosocomial infection, providing essential information for timely clinical intervention. In this study, we developed a versatile and label-free circRNA and miRNA detection platform based on a Cas13a/crRNA complex-based target recognition that triggers a primer exchange reaction (PER)-driven DNA walker. In this method, the Cas13a/crRNA complex is used to specifically identify target RNA biomarkers and to induce subsequent signal amplification strategies following cleavage of uracil (UUU, trU)-containing locked “sensing probe” based on trans-cleavage of Cas13a. Taking the merit of the high specific target recognition capability of Cas13a/crRNA, false signals or unintended circRNA degradation can be greatly avoided. The PER performed on the surface of streptavidin-coated magnetic beads (sMBs) motivated the walking of primer sequences, greatly amplifying fluorescence signals. Based on this design, the proposed method exhibits high sensitivity and precision, with detection limits as low as 0.41 fM for miRNA-21 and 3.12 fM for cZNF292. These findings suggest that this strategy could advance the identification of RNA biomarkers and hold considerable potential for future applications in diagnosing nursing infections.
研究领域:
分子诊断
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CRISPR技术
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核酸检测
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传染病监测
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生物传感器
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Li Li,Yuanqian Xiong,Yuqian Guo,Hong Duan,Yuankui Leng,Xiaolin Huang,Gang Chen,Yonghua Xiong
DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10–108 CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.
研究领域:
生物传感器
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分子诊断
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纳米技术
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食品安全
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CRISPR技术
·
分析化学
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2025
·
IF 2.6
Zixun Wang,Dong Zhang,Yuxuan Wu,Xu Ye,Yayun Qian,Fengsong Chen
The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au–S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous “hotspots”, thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm−1, a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = −3527.97 × −35369.60, R2 = 0.97767), with a LOD as low as 4.17 × 10−17 M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.
研究领域:
生物医学工程
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分子诊断
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纳米生物技术
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癌症生物标志物
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表面增强拉曼散射（SERS）
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CRISPR技术
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2025
·
IF 7.7
Zhengfang Liang,Shuwei Xiao,Junhong Li,Guoli Zuo,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision repair pathway, is frequently overexpressed in multiple cancers and has emerged as an important biomarker for early diagnosis and therapeutic response evaluation. In this work, a highly sensitive electrochemiluminescence (ECL) biosensor was developed for APE1 detection by integrating a PAM-regulated CRISPR-Cas12a activation mechanism with a multifunctional nanocomposite-modified electrode. A Ti₃C₂/AuNPs/CsPbBr₃ ternary nanocomposite was fabricated through ultrasonic assembly to provide enhanced conductivity, water stability, and probe-loading capacity. Meanwhile, a DNA duplex containing an AP site within the PAM region was engineered to regulate Cas12a activation through APE1-mediated cleavage and polymerase-assisted repair, thereby minimizing background interference and enabling highly specific signal generation. Ferrocene-labeled DNA tetrahedrons (TDNs) were further introduced as a spatially ordered probe scaffold to improve hybridization efficiency and facilitate Cas12a trans-cleavage–mediated signal release. This work presents three key innovations: (1) Construction of a Ti₃C₂/AuNPs/CsPbBr₃ nanocomposite that simultaneously enhances ECL stability, conductivity, and electron-transfer efficiency, addressing the inherent water-instability of perovskite emitters. (2) Coupling of a PAM-regulated APE1–KF repair mechanism with Cas12a activation to achieve highly specific AP-site recognition and selective signal generation. (3) Introduction of a TDN-based probe architecture that provides rigid spatial orientation and high-density probe loading, markedly improving hybridization efficiency and Cas12a trans-cleavage performance. Benefiting from these synergistic innovations, the biosensor achieves a detection limit of 6.28 × 10⁻⁸ U μL⁻¹ and a wide linear range (1 × 10⁻⁷ to 1 × 10⁻ 1 U μL⁻¹). The platform also demonstrates excellent specificity, reproducibility, and reliable performance in diluted serum, highlighting its promise for clinical and biochemical applications.
研究领域:
生物传感器
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纳米技术
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癌症诊断
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电分析化学
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分子生物学
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CRISPR技术
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产品关联
DOI
高影响因子
Biosensors-Basel
·
2025
·Vol. 16
·期 1
·17
·
IF 5.6
Zhixian Liang,Jie Zhang,Shaohui Zhang
MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO 4 3− modified single-stranded DNAs (ssDNAs); the resulting 3′ hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10 8 fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.
研究领域:
分子诊断
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生物分析化学
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CRISPR技术
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癌症研究
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核酸检测
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产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2025
·
IF 15.7
Qianqian Ying,Yongchen Chen,Luochen Shen,Yang Xu,Ruilin Tian
RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq ( SPL icing regulator i dentification through CR ISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 ( PPP1R15A ) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway. Systematic investigation of RNA splicing regulation remains challenging. Here, authors present SPLiCR-seq, a CRISPR platform enabling direct measurement of splicing outcomes for pooled perturbations. They identify GADD34 as a novel IRE1α-XBP1 regulator, with inhibition enhancing CAR-T cell activity.
研究领域:
分子生物学
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遗传学
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RNA生物学
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免疫治疗
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细胞信号转导
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CRISPR技术
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癌症研究
✓ 已验证
DOI
PubMed
高影响因子
Biosensors-Basel
·
2025
·Vol. 15
·期 8
·479
·
IF 5.6
Xiaotian Guan,Peizheng Wang,Yi Wang,Shuqing Sun
The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.
研究领域:
生物分析
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分子诊断
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CRISPR技术
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分析化学
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病毒学
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产品关联
DOI
PubMed
高影响因子
Cell Reports
·
2025
·
IF 6.9
Tianzi Wei,Jiaxuan Li,Xiang Lei,Risheng Lin,Qingyan Wu,Zhenfeng Zhang,Shimin Shuai,Ruilin Tian
Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.
研究领域:
分子生物学
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遗传学
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RNA生物学
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CRISPR技术
·
病毒学
·
基因编辑
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功能基因组学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Miao He,Jinhua Chen,MuRilege Chao,Wenting Cheng,Han Pan,Qiufeng Wang,Zhongyun Wang,Yang Xiang
mRNA vaccine had emerged as a novel and promising modality in cancer immunotherapy. As anti-tumor mRNA vaccines entered clinical trials, continuous monitoring of mRNA levels becomes particularly important. In this paper, a new method of collaborative detection of tumor mRNA vaccine by double Cas12a system based on split crRNA was created. Specifically, the target was extended based on the RCA ring template to generate RCA products and completed the first round of signal amplification; At this time, there were a lot of secondary structures in RCA products. Subsequently, the double Cas12a system with split crRNA bound to adjacent sites, reduced secondary structure of the DNA strand, stabilized the linearization state, and identified multiple sites at the same time, so as to achieve the second round of collaborative signal amplification. When there was no target in the system, the RCA reaction cannot be triggered. In this method, the entire detection process was completed by one-pot method. The detection limit was as low as 0.2pM, and it showed a good linear relationship in the range of 0–100 pM, which provided a new method for the screening and pharmacokinetics study of tumor mRNA vaccines.
研究领域:
癌症免疫治疗
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分子诊断
·
CRISPR技术
·
生物分析化学
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药代动力学
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核酸扩增
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）