Acta Pharmaceutica Sinica B
·
2023
·
IF 14.5
Yongchun Pan,Xiaowei Luan,Fei Zeng,Xuyuan Wang,Shurong Qin,Qianglan Lu,Guanzhong He,Yanfeng Gao,Xiaolian Sun,Xin Han,Bangshun He,Yujun Song
Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H 2 O 2 -mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H + and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H 2 O 2 -mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.
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ACS Nano
·
2023
·
IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
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ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Yuwan Du,Shuaifeng Ji,Qingyang Dong,Jiang Wang,Dianpeng Han,Zhixian Gao
Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans -activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy . This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.
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Nanoscale
·
2022
·Vol. 14
·期 39
·14645-14660
·
IF 8.307
Li-Min Zhai,Yan Zhao,Rui-Lin Xiao,Shi-Quan Zhang,Bao-Hua Tian,Xin-Xin Li,Rong Zhang,Ri-Sheng Ma,Hai-Xia Liang
Nuclear targeted delivery has great potential in improving the efficiency of non-viral carrier mediated genome editing. However, direct and efficient delivery of CRISPR/Cas9 plasmid into the nucleus remains a challenge. In this study, a nuclear targeted gene delivery platform based on fluorescent carbon quantum dots (CQDs) was developed. Polyethylenimine (PEI) and polyethylene glycol (PEG) synergistically passivated the surface of CQDs, providing an excitation-independent green-emitting fluorescent CQDs–PEI–PEG conjugate (CQDs–PP) with an ultra-small size and positive surface charge. Here we show that CQDs–PP could bind CRISPR/Cas9 plasmid to form a nano-complex by electrostatic attraction, which can bypass lysosomes and enter the nucleus by passive diffusion, and thereby improve the transfection efficiency. Also, CQDs–PP could deliver CRISPR/Cas9 plasmid into HeLa cells, resulting in the insertion/deletion mutation of the target EFHD1 gene. More importantly, CQDs–PP exhibited a considerably higher gene editing efficiency as well as comparable or lower cytotoxicity relative to Lipo2000 and PEI-passivated CQDs–PEI (CQDs–P). Thus, the nuclear-targeted CQDs–PP is expected to constitute an efficient CRISPR/Cas9 delivery carrier in vitro with imaging-trackable ability.
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ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xiaofen Zhao,Xiaoxiao Tian,Yuwei Wang,Linbin Li,Yan Yu,Shiqiao Zhao,Juan Zhang
Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA . After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment , direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.
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BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Hai-Yan Jia,Hong-Li Zhao,Ting Wang,Pin-Ru Chen,Bin-Cheng Yin,Bang-Ce Ye
MicroRNAs (miRNAs) play an essential role in cancer diagnosis and prognosis. Developing a new method for sensitive detection of miRNA is constantly in demand. CRISPR/Cas12a system can nonspecifically cleave single-stranded DNA after specific recognition of target DNA, showing tremendous potential in molecular diagnostics. However, CRISPR-based detection methods require synthesizing different crRNAs for detecting different targets, which limit their widespread application. Herein, we design a versatile and sensitive miRNA detection platform based on CRISPR/Cas12a system combined with a hybridization chain reaction (HCR) circuit. In this design, the HCR circuit as the signal transducer converts each miRNA into multiple DNA duplexes, which act as the activators to activate the trans -cleavage activity of Cas12a for further signal amplification . More importantly, this platform can sensitively detect different miRNAs without changing the spacer sequence of crRNA due to the fixed activators formed by HCR. In addition, the consistency between the proposed platform and RT-qPCR in miRNA detection extracted from different cell lines validated its practicability, demonstrating the potential in clinical diagnosis of cancers and monitoring therapy.
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JOURNAL OF NANOBIOTECHNOLOGY
·
2023
·Vol. 21
·期 1
·1-8
·
IF 10.2
Zheng Laibao,Jiang Yayun,Huang Fuyuan,Wu Qiaoli,Lou Yongliang
A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform’s high accuracy and sensitivity, with a limit of detection of 101 CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.
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Biology-Basel
·
2021
·Vol. 10
·期 10
·1038
·
IF 5.079
Haodong Cui,Min Jiang,Wenhua Zhou,Ming Gao,Rui He,Yifan Huang,Paul K. Chu,Xue-Feng Yu
Simple SummaryCRISPR/Cas9 system as a potential gene editing platform has been widely applied in biological engineering and disease therapies. To achieve precise gene targeting, active CRISPR/Cas9 components must be efficiently transported to targeted cells. As a simple and effective strategy, Cold Atmospheric Plasma (CAP) treatment has been demonstrated for the transmembrane delivery of various exogenous materials. In comparison with carrier-dependent delivery methods, this carrier-free platform provides a promising alternative to circumvent the obstacles of biosafety and complicated preparation processes. In this work, a CAP-based CRISPR/Cas9 carrier-free delivery platform has been established and corresponding mechanism related to efficient transportation has been explored. Briefly, the efficient production of bioactive species in culture media after CAP treatment alters cell membrane potential and permeability, which facilitates cytosolic delivery of active CRISPR/Cas9 components via passive diffusion and ATP-dependent endocytosis pathways, resulting in efficient genome editing and gene silencing. This carrier-free strategy using CAP-based transportation may also be extended to other active biomolecules in drug delivery and gene therapy.AbstractA carrier-free CRISPR/Cas9 ribonucleoprotein delivery strategy for genome editing mediated by a cold atmospheric plasma (CAP) is described. The CAP is promising in many biomedical applications due to efficient production of bioactive ionized species. The MCF-7 cancer cells after CAP exposure exhibit increased extracellular reactive oxygen and nitrogen species (RONS) and altered membrane potential and permeability. Hence, transmembrane transport of Ca2+into the cells increases and accelerates ATP hydrolysis, resulting in enhanced ATP-dependent endocytosis. Afterwards, the increased Ca2+and ATP contents promote the release of cargo into cytoplasm due to the enhanced endosomal escape. The increased membrane permeability also facilitates passive diffusion of foreign species across the membrane into the cytosol. After CAP exposure, the MCF-7 cells incubated with Cas9 ribonucleoprotein (Cas9-sgRNA complex, Cas9sg) with a size of about 15 nm show 88.9% uptake efficiency and 65.9% nuclear import efficiency via passive diffusion and ATP-dependent endocytosis pathways. The efficient transportation of active Cas9sg after the CAP treatment leads to 21.7% and 30.2% indel efficiencies in HEK293T and MCF-7 cells, respectively. This CAP-mediated transportation process provides a simple and robust alternative for the delivery of active CRISPR/Cas9 ribonucleoprotein. Additionally, the technique can be extended to other macro-biomolecules and nanomaterials to cater to different biomedical applications.Keywords:cold atmospheric plasma;CRISPR/Cas9;carrier-free transport;genome editing
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VETERINARY RESEARCH
·
2025
·Vol. 56
·期 1
·1-14
·
IF 3.5
Li Peiyao,Su Rina,Ren Ganglin,Si Hongbin,Song Xingju,Hu Dandan
The apicomplexan parasite Eimeria is the causative agent of coccidiosis, which results in enteritis in animals and imposes a significant economic burden on the farming industry. Apicomplexan Apetala2/ERF (ApiAP2) transcription factors are known to play crucial roles in various processes of Apicomplexa parasites. However, little is understood regarding the role of ApiAP2s in Eimeria species. In this study, we performed a functional characterisation of ApiAP2 through CRISPR-Cas9-mediated knockout. Our results revealed that the parasite’s invasion efficiency, total oocyst output, and virulence were significantly impaired after EtAP2-S1 depletion. RNA sequencing (RNA-Seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) analyses showed that EtAP2-S1 targets the promoters of numerous genes, and its knockout resulted in the up-regulation of 59 sag genes. Furthermore, the knockout strain exhibited significantly lower virulence while offering excellent immune protection, making it a promising candidate for a vaccine. This study demonstrates that EtAP2-S1 is a fitness-conferring gene that suppresses the expression of sag genes in E. tenella , which is the first step in developing gene-knockout vaccines to control coccidiosis.
研究领域:
分子生物学
·
寄生虫学
·
免疫学
·
兽医学
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Exploration
·
2024
·
IF 0
Yiming Zhang,Zhi Chen,Songrui Wei,Yujun Zhang,Hai Fu,Han Zhang,Defa Li,Zhongjian Xie
Real-time polymerase chain reaction (RT-PCR) remains the most prevalent molecular detection technology for sewage analysis but is plagued with numerous disadvantages, such as time consumption, high manpower requirements, and susceptibility to false negatives. In this study, an automated robot-driven photoelectrochemical (PEC) biosensing platform is constructed, that utilizes the CRISPR/Cas12a system to achieve fast, ultrasensitive, high specificity detection of biological loads in sewage. The Shennong-1 robot integrates several functional modules, involving sewage sampling and pretreatment to streamline the sewage monitoring. A screen-printed electrode is employed with a vertical graphene-based working electrode and enhanced with surface-deposited Au nanoparticles (NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the double-stranded DNA (dsDNA) anchored on Au NPs. Using the cDNA template of Omicron BA.5 spike gene as a model, the PEC biosensor demonstrates excellent analytical performance, with a lower detection limit of 2.93 × 10 2 z m and an outstanding selectivity at the level of single-base mutation recognition. Furthermore, the rapid, accurate detection of BA.5 in sewage demonstrates the feasibility of the PEC platform for sewage monitoring. In conclusion, this platform allows early detection and tracking of infectious disease outbreaks, providing timely data support for public health institutions to take appropriate prevention and control measures.
研究领域:
环境科学
·
生物传感技术
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分子生物学
·
公共卫生
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DRUG METABOLISM AND DISPOSITION
·
2019
·Vol. 47
·期 12
·1425-1432
·
IF 3.354
Hongqiu Zhou,Li Yang,Changsuo Wang,Zhiqiang Li,Zhen Ouyang,Mangting Shan,Jun Gu,Yuan Wei
Rat CYP2D1 has been considered as an ortholog of human CYP2D6. To assess the role of CYP2D1 in physiologic processes and drug metabolism, a CYP2D1-null rat model was generated with a CRISPR/Cas9 method. Seven base pairs were deleted from exon 4 of CYP2D1 of Sprague-Dawley wild-type (WT) rats. The CYP2D1-null rats were viable and showed no abnormalities in general appearance and behavior. The metabolism of venlafaxine (VLF) was further studied in CYP2D1-null rats. The Vmax and intrinsic clearance of the liver microsomes in vitro from CYP2D1-null rats were decreased (by ∼46% and ∼57% in males and ∼47% and ∼58% in females, respectively), while the Michaelis constant was increased (by ∼24% in males and ∼25% in females) compared with WT rats. In the pharmacokinetic studies, compared with WT rats, VLF in CYP2D1-null rats had significantly lower apparent total clearance and apparent volume of distribution (decreased by ∼36% and ∼48% in males and ∼23% and ∼25% in females, respectively), significantly increased area under the curve (AUC) from the time of administration to the last time point, AUC from the start of administration to the theoretical extrapolation, and Cmax (increased by ∼64%, ∼59%, and ∼26% in males and ∼43%, ∼35%, and ∼15% in females, respectively). In addition, O-desmethyl venlafaxine formation was reduced as well in CYP2D1-null rats compared with that in WT rats. Rat depression models were developed with CYP2D1-null and WT rats by feeding them separately and exposing them to chronic mild stimulation. VLF showed better efficacy in the WT depression rats compared with that in the CYP2D1-null rats. In conclusion, a CYP2D1-null rat model was successfully generated, and CYP2D1 was found to play a certain role in the metabolism and efficacy of venlafaxine.
研究领域:
药理学
·
遗传学
·
分子生物学
·
行为科学
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INDUSTRIAL CROPS AND PRODUCTS
·
2021
·
IF 5.645
Min Shi,Zhiyan Du,Qiang Hua,Guoyin Kai
Phenolic acids comprise the major bioactive compounds in Salvia miltiorrhiza , which can be increased by abscisic acid (ABA) elicitation. However, the underlying regulation mechanism is poorly understood. Here, the ABA-induced transcriptome revealed that bZIP2 was significantly induced, which expressed highly in roots. Bioinformatic analysis revealed that bZIP2 belong to the subgroup A and contained a bZIP domain together with four conservative regions (C1, C2, C3, and C4). Phenolic acid content was increased in bZIP2 knock-out lines medicated by CRISPR/Cas9 technology. In contrast, overexpressing lines produced less phenolic acids by downregulating the PAL gene. Biochemistry assays showed that bZIP2 binds to the ABRE2 element in the promoter region of the PAL gene and suppresses its expression. Besides, bZIP2 physically interacted with SnRK2.3, SnRK2.4, SnRK2.6, and SnRK2.10. Overall, these results demonstrated that bZIP2 is a negative regulator in phenolic acid biosynthesis, which provides new insights for the metabolic engineering of phenolic acids.
研究领域:
植物学
·
遗传学
·
代谢工程
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