Genes
·
2025
·Vol. 16
·期 2
·217
·
IF 2.8
Haiyan Shu,Aiping Luan,Hidayat Ullah,Junhu He,You Wang,Chengjie Chen,Qing Wei,Rulin Zhan,Shenghe Chang
Background/Objectives: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. Methods: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. Results: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5′-NGG (“N” represents any nucleobase, followed by two guanine “G”) PAM sites at the 3′ end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5′-NGG PAM site at the 3′ end. Conversely, although the target sequence with a 5′-NAG PAM site (where “N” is any nucleobase, followed by adenine “A” and guanine “G”) adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5′-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1,AcOT5,AcCSPE6,AcPKG11A), more deletions than insertions were found. Conclusions: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.
CAS / MDL:
研究领域:
植物学
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遗传学
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分子生物学
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PubMed
高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 8
·1-9
·
IF 5.3
Zhou Yanmei,Che Shengjun,Wang Zhili,Zhang Xiaoru,Yuan Xunyi
An electrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs. Graphical abstract
研究领域:
生物化学
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分子生物学
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分析化学
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生物技术
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PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2024
·
IF 4.4
Bing Fu,Meng Chen,Xianfeng Bao,Jiajie Lu,Zhiwen Zhu,Fuyao Guan,Chuyang Yan,Peize Wang,Linglin Fu,Ping Yu
Vitamin B 2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
研究领域:
生物工程
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分子生物学
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PubMed
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 4
·1-8
·
IF 5.479
Chang Weidan,Liu Weipeng,Liu Ying,Zhan Fangfang,Chen Huifang,Lei Hongtao,Liu Yingju
A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA.
Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.
研究领域:
分子生物学
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植物病理学
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纳米生物技术
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DOI
PubMed
高影响因子
Acta Pharmaceutica Sinica B
·
2024
·
IF 14.7
Xiaoyu Wu,Zijun Bai,Hui Wang,Hanqing Wang,Dahai Hou,Yunzhu Xu,Guanqun Wo,Haibo Cheng,Dongdong Sun,Weiwei Tao
Copper-based nanomaterials demonstrate promising potential in cancer therapy. Cu + efficiently triggers a Fenton-like reaction and further consumes the high level of glutathione, initiating chemical dynamic therapy (CDT) and ferroptosis. Cuproptosis, a newly identified cell death modality that represents a great prospect in cancer therapy, is activated. However, active homeostatic systems rigorously keep copper levels within cells exceptionally low, which hinders the application of cooper nanomaterials-based therapy. Herein, a novel strategy of CRISPR-Cas9 RNP nanocarrier to deliver cuprous ions and suppress the expression of copper transporter protein ATP7A for maintaining a high level of copper in cytoplasmic fluid is developed. The Cu 2 O and organosilica shell would degrade under the high level of glutathione and weak acidic environment, further releasing RNP and Cu + . The liberated Cu + triggered a Fenton-like reaction for CDT and partially transformed to Cu 2+ , consuming intracellular GSH and initiating cuproptosis and ferroptosis efficiently. Meanwhile, the release of RNP effectively reduced the expression of copper transporter ATP7A, subsequently increasing the accumulation of cooper and enhancing the efficacy of CDT, cuproptosis, and ferroptosis. Such tumor microenvironment responsive multimodal nanoplatform opens an ingenious avenue for colorectal cancer therapy based on gene editing enhanced synergistic cuproptosis/CDT/ferroptosis.
研究领域:
生物技术
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纳米技术
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肿瘤学
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基因编辑
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高影响因子
JOURNAL OF CONTROLLED RELEASE
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2025
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IF 11.5
Tong Wang,Tianyi Chen,Dazhao Li,Xiaoxing Hang,Shuangshuang Zhang,Han Yi,Tianyue Jiang,Dawei Ding,Xuenong Zhang
Cancer stem cells (CSCs) play an important role in the development of triple-negative breast cancer (TNBC), including metastasis, invasion, tumorigenicity, and drug resistance. Moreover, non-CSCs can spontaneously transform into CSCs in special tumor microenvironments, thereby leading to poor prognosis or even failed treatments. Therefore, reversing CSCs into normal tumor cells in a sustained-acting manner is a promising strategy. It has been reported that down-regulation of FBXO44 protein expression inhibits tumor cell stemness. Moreover, CRISPR/Cas9 technology, a well-known precise gene editing tool, was adopted to permanently block FBXO44 within the genome upon its successful implementation. Given this, a core-shell nanoparticle (NP) consisting of amphiphilic polymer core and crosslinked-hyaluronic acid shell (nDOX-PL/pFBXO44 NPs) is developed in this work to concurrently deliver FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and doxorubicin (DOX) for combinational CSC reprogramming and chemotherapy of TNBC, which exhibits tumor cell targeting, endosomal escape, and reduction responsiveness to release DOX and plasmids in the cytoplasma. CRISPR/Cas9-mediated downregulation of FBXO44 expression could convert CSC into normal tumor cells, and effectively inhibit tumor growth without obvious side effects in vivo after combining with chemotherapy. In summary, we developed an intelligent system to co-deliver genetic and hydrophobic drugs, achieving effective cancer stemness reversal and synergistic suppression of contractable TNBC.
研究领域:
肿瘤学
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基因编辑
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纳米医学
·
药理学
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高影响因子
JOURNAL OF COLLOID AND INTERFACE SCIENCE
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2025
·
IF 9.7
Shi-Cheng Tian,Xun-Huan Song,Ke-Ke Feng,Cheng-Lei Li,Yi-Fan Tu,Yong-Shan Hu,Jing-Wei Shao
Photodynamic therapy (PDT) has arisen as a promising method due to its spatiotemporal precision and minimal invasiveness. It encounters significant obstacles in solid tumors due to hypoxia-induced therapeutic resistance and the self-protective mechanisms of cancer cells facilitated by MutT homolog 1 (MTH1), an enzyme involved in oxidative damage repair. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on hollow mesoporous manganese dioxide (H-MnO 2 ) for PDT. This platform utilizes H-MnO 2 to produce oxygen (O 2 ) through the decomposition of hydrogen peroxide (H 2 O 2 ) in TME, thereby mitigating hypoxia and enhancing reactive oxygen species (ROS) generation. The high concentration of glutathione (GSH) and hyaluronidase (HAase) in TME induces the release of CRISPR/Cas9 ribonucleoproteins (RNP) to target the MTH1 gene, thereby impairs oxidative damage repair pathways and amplifys ROS-mediated cytotoxicity. The released Mn 2+ ions function as immunomodulatory agents, activate innate immune responses via stimulating STING signal pathway. In vitro , IHMRH NPs markedly increased intracellular O 2 levels, ROS production, lipid peroxidation and DNA damage, leading to tumor cell death, immune activation, and effective gene editing. In vivo , the nanoplatform suppressed tumor growth, diminished MTH1 gene expression, stimulated dendritic cell (DC) maturation through immunogenic cell death (ICD). This multimodal nanosystem may amplifies oxidative stress, collaborates with innate and adaptive immune activation to surpass the constraints of traditional PDT. The research presents a novel framework for cancer combination therapy by systematically integrating nanotechnology with precision gene editing.
研究领域:
生物医学工程
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肿瘤学
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基因治疗
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免疫学
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高影响因子
INTERNATIONAL JOURNAL OF PHARMACEUTICS
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2024
·
IF 5.3
Qian Sun,Hongqian Zhang,Feng Ding,Xue Gao,Zongwei Zhu,Chuanxu Yang
CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.
研究领域:
分子生物学
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基因治疗
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纳米医学
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PubMed
高影响因子
npj Precision Oncology
·
2025
·Vol. 9
·期 1
·1-21
·
IF 8
Li Xiaoli,Wang Jiashuo,Guo Jiaxing,Zhang Ming
Cisplatin resistance is a major challenge in laryngeal cancer treatment. Tumor necrosis factor-alpha-induced protein 2 (TNFAIP2) has been implicated in chemoresistance, though its role in laryngeal cancer remains unclear. We identified TNFAIP2 as a key gene associated with cisplatin resistance and developed a second near-infrared (NIR-II) light-responsive CRISPR-Cas9 nanosystem (APC@RBCs) to target it. In cisplatin-resistant Tu177/CDDP cells, light-activated knockout of TNFAIP2 significantly increased cisplatin sensitivity (IC50 reduced from 12.55 to 4.37 µg/mL). Mechanistic studies revealed that TNFAIP2 modulates cisplatin resistance via the NRF2 pathway, affecting oxidative stress response and epithelial-mesenchymal transition. TNFAIP2 deletion also suppressed cancer cell migration and invasion. Our results establish TNFAIP2 as a critical mediator of chemoresistance and demonstrate the potential of APC@RBCs as a precise, non-invasive gene therapy platform. This strategy offers a promising approach to overcoming cisplatin resistance and improving treatment outcomes in laryngeal cancer patients.
研究领域:
肿瘤学
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分子生物学
·
药理学
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纳米技术
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基因工程
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产品关联
DOI
PubMed
高影响因子
CANCER RESEARCH
·
2025
·
IF 16.6
Shang Yan,Li Shuaihu,Xu Tian,Sun Yihua,Li Fei
The CRISPR/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the VL-AdCre system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized CRISPR/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.
研究领域:
免疫学
·
肿瘤学
·
分子生物学
·
基因编辑
·
转化医学
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产品关联
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PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2020
·Vol. 48
·期 20
·11773-11784
·
IF 11.502
Lin Jiao,Liu Yan,Lai Peidong,Ye Huixia,Xu Liang
A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.
研究领域:
合成生物学
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分子遗传学
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基因工程
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PubMed
高影响因子
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.
研究领域:
遗传学
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分子生物学
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生物医学工程
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纳米技术
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DOI
PubMed
高影响因子
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