CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles.
CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles.
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CRISPR/Cas 13 a在肿瘤细胞外囊泡中的microRNA检测
DOI：
10.1002/advs.202301766
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发表时间：
2023-08
期刊：
影响因子：
15.1
通讯作者：
Im, Hyungsoon
中科院分区：
文献类型：
--
作者：
Hong, Jae-Sang;Son, Taehwang;Castro, Cesar M. M.;Im, Hyungsoon
细胞外囊泡（EV）中的微小RNA（miRNAs）在癌症的发生和发展中起着重要作用。EV miRNA的定量测量对于癌症诊断和纵向监测至关重要。然而，传统的基于PCR的方法需要多步程序，并且仍然是批量分析。在这里，作者介绍了一种使用CRISPR/Cas 13 a传感系统的无扩增和无提取EV miRNA检测方法。CRISPR/Cas 13 a传感组件封装在脂质体中，并通过脂质体-EV融合将其递送到EV中。这允许使用1 × 108 EV准确定量特定miRNA阳性EV计数。作者表明，卵巢癌EV中miR-21 - 5 p阳性EV计数在2%-10%的范围内，显著高于良性细胞的阳性EV计数（<0.65%）。结果表明，批量分析与金标准方法RT-qPCR之间具有良好的相关性。作者还通过捕获EpCAM阳性EV并定量亚群中的miR-21 - 5 p阳性EV，证明了肿瘤来源EV中的多重蛋白质-miRNA分析，其显示癌症患者血浆中的计数显著高于健康对照。所开发的EV miRNA传感系统提供了在不提取RNA的情况下在完整EV中的特异性miRNA检测方法，并开辟了用于蛋白质和RNA标记物的多重单EV分析的可能性。报道了一种基于CRISPR/Cas 13 a技术的无需扩增和提取的细胞外囊泡（EV）miRNA检测方法。CRISPR/Cas 13 a传感组件封装在脂质体中，并通过脂质体-EV融合将其递送到EV中。新方法能够在完整EV中进行多重蛋白质和RNA分析，定量人血浆样本中特异性miRNA阳性肿瘤衍生EV，以实现更准确的癌症诊断。
MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR‐based methods, however, require multi‐step procedures and remain as bulk analysis. Here, the authors introduce an amplification‐free and extraction‐free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome‐EV fusion. This allows for accurately quantify specific miRNA‐positive EV counts using 1 × 108 EVs. The authors show that miR‐21‐5p‐positive EV counts are in the range of 2%–10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold‐standard method, RT‐qPCR. The authors also demonstrate multiplexed protein‐miRNA analysis in tumor‐derived EVs by capturing EpCAM‐positive EVs and quantifying miR‐21‐5p‐positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers. An amplification‐free and extraction‐free extracellular vesicle (EV) miRNA detection method using CRISPR/Cas13a technology is reported. The CRISPR/Cas13a sensing components are encapsulated in liposomes and deliver them into EVs through liposome‐EV fusion. The new approach enables multiplexed protein and RNA analysis in intact EVs, quantifying specific miRNA‐positive tumor‐derived EVs in human plasma samples for more accurate cancer diagnosis.
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影响因子：
10
作者：
Al Sulaiman, Dana;Juthani, Nidhi;Doyle, Patrick S.
通讯作者：
Doyle, Patrick S.
CRISPR-C2C2的两种不同的RNase活性启用了指导RNA处理和RNA检测。
影响因子：
64.8
作者：
East-Seletsky A;O'Connell MR;Knight SC;Burstein D;Cate JH;Tjian R;Doudna JA
通讯作者：
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影响因子：
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通讯作者：
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影响因子：
16.8
作者：
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通讯作者：
Doudna JA
影响因子：
11.2
作者：
Ko, Jina;Bhagwat, Neha;Issadore, David
通讯作者：
Issadore, David