Biodog 发表于 2017-4-14 10:55:01

Science:张锋团队今日带来CRISPR重磅新应用,核酸检测灵敏...

本帖最后由 Biodog 于 2017-4-14 11:07 编辑

今日,知名华人科学家张锋教授与同事在《科学》杂志上发表论文,介绍了一种全新的CRISPR应用工具,用以检测核酸。这一工具能快速检测RNA或DNA分子,灵敏度甚至有望检测出单个核酸。这一重磅研究对于科研与全球公共卫生有着极大的影响。

早在2010年,科学家们就意识到,CRISPR平台也许能被用来检测病毒的核酸。但在当时,它的基因编辑潜力过于出众,光芒在一定程度上掩盖了其他领域应用的可能性。直到CRISPR-Cas9基因编辑技术趋于完善之时,科研人员才有余力检视它在病毒检测上的潜力。
科学家们在今日发表的这个系统与CRISPR-Cas9系统有着本质上的差异。它由向导RNA(gRNA)和Cas13a蛋白等部分组成。向导RNA能带领Cas13a蛋白识别并剪开带有特异序列的RNA。然而Cas13a有一个奇怪的特点,当它切开特异的RNA后,就无法管住自己的“双手”,开始疯狂切开它能遇到的任何RNA。利用这一特性,张锋教授与James Collins教授在这套系统里塞入了一种带有RNA的荧光标志物。平时它不会发出荧光,但一旦Cas13a到处“搞事情”,这种荧光标志物的RNA就会被切开,让它发出明亮的光芒。
▲这项全新CRISPR应用的原理(图片来源:《科学》)
也就是说,只要设计好合适的向导RNA,并提供Cas13a与荧光标志物,那么一旦这套系统识别到匹配的RNA序列,就会发出荧光,告诉研究人员“我们找到了!”
这套系统的灵敏度达到了50fM(1fM为10的负15次方摩尔每升)。
尽管和之前的一些研究相比,它的灵敏度提高了数百万倍,但研究人员依然没有满足。他们想要把灵敏度进一步提高,增加到阿摩尔级(aM,10的负18次方摩尔每升)。为了达到这一目标,他们又用上了“等温扩增”(isothermal amplification)技术,进一步降低了对初始核酸浓度的需求。
在后续的实验中,研究人员发现,使用“等温扩增”技术的这套CRISPR系统无论是对RNA还是DNA,灵敏度都达到了单分子级。
这种不遗漏任何线索的特性让研究人员想到了福尔摩斯(Sherlock Holmes)。于是他们杜撰了一个名字,把这套系统称为SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing)。
这套系统开发出来可不是为了好玩。研究人员相信,它能用于各种疾病的检测。首先,研究人员在不同的病毒上测试了它的特异性。实验表明,它能很好地区分两种很类似的寨卡病毒株。这对于准确诊断病情,有效预防疫情爆发而言,有着现实的应用价值。
随后,研究人员又开始将它用于检测癌症突变。在癌症的早期诊断领域,液体活检是一大热门——科学家们希望从血液中分离出专属于癌细胞的突变,从而得知癌症是否开始发作。然而,由于血液中有着大量的非癌细胞DNA,这一检测过程也是困难无比。在今日发表的论文中,研究团队使用现成的样本,来模拟患者的血液。而实验表明,SHERLOCK同样能以阿摩尔级的灵敏度,识别出肿瘤特有的突变。

▲在人类基因组中,这套系统能轻松检测出不同的SNP(图片来源:《科学》)
类似的应用还有很多。比如,研究人员用这套系统,成功地鉴定出了人类基因组中的多个单核苷酸多态性(SNP),并能分析出每一个人的特定SNP是纯合还是杂合。在这个基因组的时代,SHERLOCK能得到很广泛的应用。
“能将Cas13a应用到灵敏度如此非同寻常的工具中,我感到非常激动。无论对科学还是临床医学,它都是很强大的工具。”张锋教授说。
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▲Cas13a蛋白是这套系统的关键(图片来源:《科学》)
“我们现在能有效地制作针对任何核酸的探测器,在诊断或研究领域的应用上,这项工具无比强大,”该研究的另一名资深作者James Collins教授说道:“这项工具能在患者的血液中寻找到极少量的肿瘤DNA,帮助研究人员理解肿瘤的突变如何随着时间推移发生改变。另外在公共卫生领域,它也能帮助研究者监控耐药菌的发生频率。科学上它有无限让人激动的可能。”Nucleic acid detection with CRISPR-Cas13a/C2c2Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse 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 SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.

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