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Nucleic acid detection with CRISPR-Cas13a/C2c2
Science ( IF 56.9 ) Pub Date : 2017-04-13 , DOI: 10.1126/science.aam9321 Jonathan S. Gootenberg 1, 2, 3, 4, 5 , Omar O. Abudayyeh 1, 2, 3, 4, 6 , Jeong Wook Lee 7 , Patrick Essletzbichler 1, 2, 3, 4 , Aaron J. Dy 1, 4, 8 , Julia Joung 1, 2, 3, 4 , Vanessa Verdine 1, 2, 3, 4 , Nina Donghia 7 , Nichole M. Daringer 8 , Catherine A. Freije 1, 9 , Cameron Myhrvold 1, 9 , Roby P. Bhattacharyya 1 , Jonathan Livny 1 , Aviv Regev 1, 10 , Eugene V. Koonin 11 , Deborah T. Hung 1 , Pardis C. Sabeti 1, 9, 12, 13 , James J. Collins 1, 4, 6, 7, 8 , Feng Zhang 1, 2, 3, 4
Science ( IF 56.9 ) Pub Date : 2017-04-13 , DOI: 10.1126/science.aam9321 Jonathan S. Gootenberg 1, 2, 3, 4, 5 , Omar O. Abudayyeh 1, 2, 3, 4, 6 , Jeong Wook Lee 7 , Patrick Essletzbichler 1, 2, 3, 4 , Aaron J. Dy 1, 4, 8 , Julia Joung 1, 2, 3, 4 , Vanessa Verdine 1, 2, 3, 4 , Nina Donghia 7 , Nichole M. Daringer 8 , Catherine A. Freije 1, 9 , Cameron Myhrvold 1, 9 , Roby P. Bhattacharyya 1 , Jonathan Livny 1 , Aviv Regev 1, 10 , Eugene V. Koonin 11 , Deborah T. Hung 1 , Pardis C. Sabeti 1, 9, 12, 13 , James J. Collins 1, 4, 6, 7, 8 , Feng Zhang 1, 2, 3, 4
Affiliation
Sensitive and specific CRISPR diagnostics Methods are needed that can easily detect nucleic acids that signal the presence of pathogens, even at very low levels. Gootenberg et al. combined the allele-specific sensing ability of CRISPR-Cas13a with recombinase polymerase amplification methods to detect specific RNA and DNA sequences. The method successfully detected attomolar levels of Zika virus, as well as the presence of pathogenic bacteria. It could also be used to perform human genotyping from cell-free DNA. Science, this issue p. 438 An ortholog of CRISPR-Cas13a/C2c2 can be used as a highly sensitive detector of specific RNA and DNA sequences. Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
中文翻译:
使用 CRISPR-Cas13a/C2c2 进行核酸检测
需要敏感和特异的 CRISPR 诊断方法,即使在非常低的水平下,也可以轻松检测发出病原体存在信号的核酸。古腾伯格等人。将 CRISPR-Cas13a 的等位基因特异性传感能力与重组酶聚合酶扩增方法相结合,以检测特定的 RNA 和 DNA 序列。该方法成功检测到寨卡病毒的阿托摩尔水平,以及病原菌的存在。它还可用于从无细胞 DNA 进行人类基因分型。科学,这个问题 p。438 CRISPR-Cas13a/C2c2 的直向同源物可用作特定 RNA 和 DNA 序列的高灵敏度检测器。快速、廉价和灵敏的核酸检测可能有助于床旁病原体检测、基因分型和疾病监测。在 RNA 引导下,RNA 靶向成簇的规则间隔短回文重复序列 (CRISPR) 效应器 Cas13a(以前称为 C2c2)在目标识别时表现出混杂核糖核酸酶活性的“附带效应”。我们将 Cas13a 的附带效应与等温扩增相结合,以建立基于 CRISPR 的诊断 (CRISPR-Dx),提供具有阿托摩尔灵敏度和单碱基错配特异性的快速 DNA 或 RNA 检测。我们使用这种基于 Cas13a 的分子检测平台,称为特定高灵敏度酶报告基因解锁 (SHERLOCK),检测寨卡病毒和登革热病毒的特定菌株,区分致病细菌,对人类 DNA 进行基因分型,并识别无细胞肿瘤 DNA 中的突变。此外,
更新日期:2017-04-13
中文翻译:
使用 CRISPR-Cas13a/C2c2 进行核酸检测
需要敏感和特异的 CRISPR 诊断方法,即使在非常低的水平下,也可以轻松检测发出病原体存在信号的核酸。古腾伯格等人。将 CRISPR-Cas13a 的等位基因特异性传感能力与重组酶聚合酶扩增方法相结合,以检测特定的 RNA 和 DNA 序列。该方法成功检测到寨卡病毒的阿托摩尔水平,以及病原菌的存在。它还可用于从无细胞 DNA 进行人类基因分型。科学,这个问题 p。438 CRISPR-Cas13a/C2c2 的直向同源物可用作特定 RNA 和 DNA 序列的高灵敏度检测器。快速、廉价和灵敏的核酸检测可能有助于床旁病原体检测、基因分型和疾病监测。在 RNA 引导下,RNA 靶向成簇的规则间隔短回文重复序列 (CRISPR) 效应器 Cas13a(以前称为 C2c2)在目标识别时表现出混杂核糖核酸酶活性的“附带效应”。我们将 Cas13a 的附带效应与等温扩增相结合,以建立基于 CRISPR 的诊断 (CRISPR-Dx),提供具有阿托摩尔灵敏度和单碱基错配特异性的快速 DNA 或 RNA 检测。我们使用这种基于 Cas13a 的分子检测平台,称为特定高灵敏度酶报告基因解锁 (SHERLOCK),检测寨卡病毒和登革热病毒的特定菌株,区分致病细菌,对人类 DNA 进行基因分型,并识别无细胞肿瘤 DNA 中的突变。此外,