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Cell: 找到魔剪CRISPR系统的广谱抑制剂

2017-08-28 佚名 生物探索

8月24日,CRISPR先驱Jennifer Doudna教授团队在Cell杂志发表了一项重要成果。他们找到了魔剪CRISPR系统的广谱抑制剂。据不完全统计,这是自去年12月首篇关于CRISPR关闭开关的论文发表后的第4篇相关Cell论文。这些关闭开关的发现势必将帮助提高CRISPR的安全性,并通过降低脱靶效应来改善基因编辑的精准度。

2012年8月,加州大学伯克利分校的Jennifer Doudna教授与现任Max Planck研究所感染生物学部主任的Emmanuelle Charpentier教授在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。这篇论文首次揭示了CRISPR/Cas这一天然免疫系统的基因组编辑功能。


从左至右:Jennifer Doudna、张锋、Emmanuelle Charpentier(图片来源:网络)

半年后(2013年2月),Broad研究所的张锋教授带领的研究小组在Science杂志上发表了另一篇题为“Multiplex Genome Engineering Using CRISPR/Cas Systems”的论文,首次证实了CRISPR技术能够编辑人类细胞的基因组。

这两篇关键的论文开启了CRISPR技术在生命科学领域的风靡生涯。这一被誉为“魔剪”的基因编辑技术被全球的科学家们应用到各种各样的研究中。其中,最引人注目的一个领域是将这一“改写生命的神笔”用到人类疾病的治疗中。本月,发表在Nature杂志上题为“Correction of a pathogenic gene mutation in human embryos”论文中,科学家第一次成功利用CRISPR-Cas9系统在人类早期胚胎中对导致肥厚型心肌病的基因突变进行了安全修复。


图片来源:eGenesis

发表Science杂志上题为“Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9”的研究中,科学家们利用CRISPR技术解决了猪器官异种移植的一个关键难题,收获世界首批内源性逆转录病毒基因失活的幼猪。

事实上,近几年,与CRISPR治疗疾病相关研究成果可谓是层出不穷。不过,尽管大家对这一基因编辑技术的疾病治疗潜力充满了期待,但是它的脱靶效应(简单来说,就是编辑了“预想之外”的基因,带来各种不可控的后果)和安全性等问题却是不容忽视的。

如何能够更好的控制CRISPR系统是科学家们正在积极研究的一个方向。这其中就包括了CRISPR先驱Doudna教授的团队。8月24日,“女神”的团队在Cell杂志上发表了一篇题为“A Broad-Spectrum Inhibitor of CRISPR-Cas9”的论文。这次,他们找到了CRISPR-Cas9系统的一种广谱抑制剂,也就是“关闭开关”。

据不完全统计,这已经是自去年12月以来关于CRISPR“关闭开关”的第4篇Cell论文了。在正式介绍Doudna教授团队的新成果前,我们先来回顾一下其他科学家团队的前期成果。

Cell 论文一:Naturally Occurring Off-Switches for CRISPR-Cas9

CRISPR/Cas9系统的首个“关闭开关”



图片来源:Cell

2016年12月15日发表的这篇论文中,来自多伦多大学和马萨诸塞大学的科学家们首次发现了CRISPR/Cas9的“关闭开关”。研究小组鉴定出了3个天然存在的、能够抑制Cas9酶的蛋白质家族。在CRISPR/Cas9系统中,Cas9酶发挥着剪断目标DNA序列的功能,而这些称为anti-CRISPR的蛋白质具有阻断DNA切割的能力。研究还证实,这些anti-CRISPRs能够被用作人类细胞基因编辑的有效抑制剂。

Cell 论文二:Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

找到常用Cas9酶的抑制剂

不到一个月后(2017年1月12日),来自加州大学旧金山分校(UCSF)的科学家们在Cell杂志发表了关于关闭开关的第二篇重要论文。

具体来说,研究人员在李斯特菌中找到了多种称为anti-CRISPRs的抑制剂。其中,AcrIIA2和AcrIIA4两个抑制剂能够阻断来自酿脓链球菌的Cas9酶或SpyCas9酶活性。目前,Cas9酶被频繁用于基因组编辑中,而SpyCas9是Cas9的变体,现在被世界大多数实验室使用。

需要指出的一点是,“论文一”中发现的关闭开关能特异性抑制脑膜炎奈瑟氏球菌的CRISPR-Cas9系统,绑定NmeCas9酶(N. meningitidis Cas9),但不能作用于现在被广泛使用的来自酿脓链球菌的Cas9酶。


图片来源:Cell

科学家们认为,anti-CRISPR蛋白的发现将成为研究和治疗复杂和多基因疾病的关键。在同期的报道中,研究人员表示,他们下一步的研究计划是调查这些CRISPR抑制剂是否能够在人类细胞中通过降低脱靶效应改善基因编辑的精准度。同时,他们也非常想弄清楚,这些抑制蛋白是如何阻断Cas9基因编辑活性的。

Cell 论文三:Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex

CRISPR“关闭开关”如何起作用

3月23日,发表在Cell杂志上的另一篇论文回答了“论文二”作者的一个疑问,即这些关闭开关究竟是如何抑制CRISPR系统的。


This image shows how the CRISPR surveillance complex is disabled by two copies of anti-CRISPR protein AcrF1 (red) and one AcrF2 (light green). These anti-CRISPRs block access to the CRISPR RNA (green tube) preventing the surveillance complex from scanning and targeting invading viral DNA for destruction (Image from Lander Lab). Credit: The Scripps Research Institute

具体来说,在这一研究中,由Scripps研究所的生物学家Gabriel C. Lander领导的研究小组第一次解决了病毒的anti-CRISPR蛋白和细菌的CRISPR复合物结合的结构,准确地揭示了病毒是如何让细菌防御系统失效的。

这篇Cell论文中涉及的两个anti-CRISPR蛋白是AcrF1 和AcrF2。而仅仅一个月后,关于“论文二”中的anti-CRISPR蛋白AcrIIA2和AcrIIA4是如何抑制SpyCas9活性的论文也发表在了Nature杂志上。(论文标题:Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein;发表日期:4月27日)这一成果来自哈尔滨工业大学生命学院黄志伟教授课题组。

Cell 论文四:A Broad-Spectrum Inhibitor of CRISPR-Cas9

女神团队发现广谱CRISPR-Cas9抑制剂


图片来源:Cell

最后,让我们来看看Doudna教授团队最新发表在Cell杂志上的这篇论文。这一研究发现,AcrIIC1 和AcrIIC3两种anti-CRISPR蛋白能够通过不同的策略来抑制Cas9酶。研究证实,AcrIIC1是一种广谱的Cas9抑制剂,它可以通过直接结合到Cas9酶保守的HNH催化域来阻止多种不同Cas9同源(multiple divergent Cas9 orthologs)的DNA切割。AcrIIC1-Cas9 HNH域复合物(AcrIIC1-Cas9 HNH domain complex)的晶体结构揭示了AcrIIC1是如何限制Cas9酶的。相比而言,AcrIIC3能阻断单个Cas9同源(Cas9 ortholog)的活性,在阻止Cas9绑定靶向DNA时诱导了Cas9的二聚化。



总结

CRISPR的强大让一些研究者和公众担心它会被不合理的使用,带来危险。这些关闭开关的发现为阻止不法的、失控的CRISPR应用提供了手段,将帮助提高CRISPR研究的安全性。

Erik J. Sontheimer博士说:“一旦基因编辑系统进入细胞后,科学家们缺乏一个可靠的方法来关闭Cas9的活性。如果我们能够在正确的基因编辑完成后打开一个‘关闭开关’,问题就很容易解决了。“关闭开关”的发现将使研究人员能够更加自信的使用这一工具。”

原始出处:

[1] A Pawluk, N Amrani, Y Zhang, et.al. Naturally Occurring Off-Switches for CRISPR-Cas9. Cell, 2016

[2] BJ Rauch, MR Silvis, JF Hultquist, et.al. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell 2017 


[4] Lucas B. Harrington, Kevin W. Doxzen, Enbo Ma, et.al. A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell

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    2018-02-02 jklm09
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    2017-10-08 维他命
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    2017-08-29 yuandd

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