大牛David Liu最新Nature:“升级版”Cas9,让基因编辑更灵活精确
2018/03/06
近期,就在张锋、Jennifer Doudna争先发表最新成果的同时,同样在CRISPR领域有“大神”之称的David R. Liu教授也在Nature期刊发表了一项突破性成果。他带领团队获得了一种“升级版”的Cas9酶,并证实可以编辑更多的基因位点。


David Liu(Credit: Martin Adolfsson)

自2012年被证实具有基因组编辑功能以来,CRISPR/Cas9已经成为深受实验室喜欢的工具,并在纠正致病突变、寻找癌症免疫疗法的必需基因、解决器官异种移植关键难题等领域创造了多个突破和成果。但是该技术仍然存在一些局限性,例如“编辑范围有限”、“脱靶效应”等。

很多研究团队一直试图优化这一工具。哈佛大学/Broad研究所的化学生物学家David R. Liu就是其中一员。2月28日,其团队在《Nature》期刊在线发表了题为“Evolved Cas9 variants with broad PAM compatibility and high DNA specificity”的文章。他们研发出一种Cas9酶的变体,是基础版酿脓链球菌Cas9酶(SpCas9)的“升级版”。


doi:10.1038/nature26155

David R. Liu将其命名为“xCas9”,并证实其在基因编辑时更灵活、更精确——可以靶向更多的基因位点,并减少“错误编辑”的风险。

CRISPR系统的关键在于Cas9酶在引导RNA(gRNA)的指引下负责切割特定的DNA序列。其中,spCas9的识别依赖于特定的PAM序列(位于编辑位点附近),该序列的一个末端是由特定的三碱基排列组成的。这种排列简称为N,由组成DNA的任一种碱基(A、G、C、T)再加上2个鸟嘌呤(G)组成。反观人类基因组,只有1/16的区域含有此类PAM序列(NGG)。这无疑限制了基因编辑的范围。

The genome editor CRISPR cuts DNA with help from a guide RNA (green and red) and a Cas9 enzyme (outline) that latches onto a three-base sequence (yellow). KC ROEYER/UNIVERSITY OF CALIFORNIA, BERKELEY

为了打破这一局限,David R. Liu实验室尝试改造,使其能够在多种PAM序列旁进行切割。通过phage-assisted continuous evolution(PACE)方法,最终他们获得了xCas9——其可以广泛识别多种PAM序列,包括NG、GAA和GAT,编辑范围至少增加了4倍,可以靶向编辑基因组的1/4区域。

研究团队对xCas9进行多种测试,验证其与“base editors”工具结合时置换单个剪辑的能力。让他们惊喜的是,相比于Cas9,xCas9错误编辑的概率降低了。

David R. Liu强调,对于xCas9酶的潜能,依然需要时间验证。他和团队目前只测试了几十个位点,相比于xCas9成千上万的目标只是“冰山一角”。“我并不能确定,xCas9一定优于spCas9。所以我希望更多的人来检测它,因为我想知道答案。” David R. Liu表示道。

参考资料:

Upgrade makes genome editor CRISPR more muscular, precise

Powerful enzyme could make CRISPR gene-editing more versatile

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  • Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

    A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT. The PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.

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