Science:与张锋同一天!女神也上Science:CRISPR变身诊断工具,可检测HPV

2018-02-28 Chen 生物探索

俗话说,一时瑜亮。在CRISPR领域,Broad研究所的张锋与加州大学伯克利分校的Jennifer Doudna是众所周知的竞争对手。2月15日,就在张锋团队发布新成果的同时, “女神”也“出手了”!

俗话说,一时瑜亮。在CRISPR领域,Broad研究所的张锋与加州大学伯克利分校的Jennifer Doudna是众所周知的竞争对手。2月15日,就在张锋团队发布新成果的同时, “女神”也“出手了”!

早在2012年8月,Doudna教授与现任Max Planck研究所感染生物学部主任的Emmanuelle Charpentier在Science杂志上发表了一篇题为“A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity”的重要成果,首次揭示了CRISPR/Cas这一天然免疫系统的基因组编辑功能。

In the genome editing technology known as CRISPR-Cas9, RNA (blue) guides the protein Cas9 (large bumpy structure) to a target site in DNA (red). Cas9 unwinds the DNA double helix and acts as molecular scissors, snipping both strands of DNA.

之后,以CRISPR-Cas9为代表的新型基因编辑技术迅速风靡科研圈,革新了生物医学研究,加速了疾病成因的探索,并引发了许多潜在的新疗法。与此同时,CRISPR家族也不断扩大,很多“新成员”浮出水面,其中就包括了CRISPR-Cas12a。

1关键酶——Cas12a

与大家熟知的Cas9酶类似,Cas12a可用于切割目标DNA。该蛋白最初被称为Cpf1,于2015年由张锋团队在一篇题为“Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System”的Cell论文中首次提出。

学界认为,Cas12a是基因切割工具箱的一个重要补充,能够在Cas9不能作用的位置切割双链DNA;同时,由于Cas12a切割DNA后留下的是不规则的边缘(ragged edges),这可能使在切割处插入一个新的基因更容易。

不过,Doudna教授的团队发现,当Cas12a结合并切割了目标双链DNA时,它会在试管中出人意料地对所有的单链DNA发动任意切割。

值得庆幸的是,由于细胞中大部分DNA是以双链螺旋的形式存在,因此,这一发现对于Cas12a的基因编辑功能并不一定是个问题。相反,Cas12a对单链DNA的这种“任意切割”还给科学家们带来了惊喜:允许他们将一个单链“报告”分子与Cas12a蛋白进行联用,当Cas12a发现它的目标后,报告分子会产生一种清晰的荧光信号。

2快速、简便的诊断系统——DETECTR

基于这一思路,Doudna教授团队开发出一种被称为“DETECTR”(DNA Endonuclease Targeted CRISPR Trans Reporter)的诊断系统,可用于对临床样本中的少量DNA进行快速、简便地即时检测。相关成果以“CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity”为题发表在Science杂志上。

Infographic by the Howard Hughes Medical Institute

具体来说,DETECTR是在一个单一反应中添加所有试剂,包括CRISPR-Cas12a和它的向导RNA、荧光报告分子以及一种叫做RPA(recombinase polymerase amplification,类似于PCR)的等温扩增系统。当加热到体温时,RPA会快速增加目标DNA的拷贝数,使Cas12a更容易找到并切割它,然后任意切割附近单链DNA,从而让报告分子发出荧光(具体发光机制见上图)。

基于CRISPR-Cas12a的DETECTR系统能够分析细胞、血液、唾液、尿液和粪便,以检测基因突变、癌症和抗生素耐药性,以及诊断细菌和病毒感染

研究中,科学家们利用包含人乳头瘤病毒(human papilloma virus,HPV)的患者样本对DETECTR进行了测试。结果显示,利用DETECTR,他们能够在感染多种不同HPV类型的样本中准确测定出高风险的HPV 类型:HPV16和HPV18。

论文的共同第一作者Janice S. Chen认为,Cas12a可作为一种强有力的工具,用于从各种各样的来源中检测DNA。DETECTR这项技术可能适用于任何基于DNA检测的即时诊断情况,包括癌症和传染病。

3再次验证CRISPR酶的检测功能

事实上,Cas12a与CRISPR酶家族的另一个蛋白Cas13a活性类似。也正是利用后者,张锋团队在同日发表在Science论文中提出了升级版的诊断工具SHERLOCK。详细阅读《张锋最新Science!CRISPR新功能升级,可用于肿瘤DNA等多种核酸检测》一文。

Cas13a于2016年6月由张锋团队首次发现;3个月后,女神研究组就证实了该酶的“collateral cleavage”活性(Cas13a在切割了它的意向RNA目标后依然能够保持“活跃”,继续爆发性式地切割其他非目标RNA)可用于RNA检测。不多久(2017年4月),张锋团队也开发出了基于Cas13a的初级版SHERLOCK。两大阵营在这一研究方向的竞争可谓非常激烈。

Doudna教授说:“现在,我们仍着迷于细菌CRISPR系统的功能,以及如何借助对相关机制的理解来产生新的技术。”

此次,双方于同日在Science发表新成果,不管是杂志刻意安排,还是一种巧合,都再次证实了CRISPR酶用于核酸检测的实力。或许,除了基因编辑,CRISPR还能带给我们带来更多、更大的惊喜!

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    2018-04-17 wgsun
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    2018-07-03 固块

    学习了

    0

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    2018-03-02 yuandd
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    2018-03-02 jichang
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    2018-02-28 祝您健康

    学习了.更幸运的事文中有同名同姓!谢谢

    0

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