CRISPR/Cas9-mediated genome editing in a reef-building coral
CRISPR/Cas9-mediated genome editing in a reef-building coral
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DOI：
10.1073/pnas.1722151115
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发表时间：
2018-05-15
影响因子：
11.1
通讯作者：
Matz, Mikhail V.
中科院分区：
文献类型：
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作者：
Clevesa, Phillip A.;Strader, Marie E.;Matz, Mikhail V.
造礁珊瑚是极其重要的物种，受到包括气候变化在内的人为压力的威胁。为了了解珊瑚对压力的反应及其生物学的其他方面，进行了许多基因组和转录组学研究，产生了关于特定基因和分子途径作用的各种假设。然而，由于缺乏珊瑚的遗传工具，通常不可能严格测试这些假设。在这里，我们展示了在珊瑚Acropora millepora中使用CRISPR/Cas9系统进行的高效基因组编辑。我们靶向编码成纤维细胞生长因子1a（FGF 1a）、绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的基因。在将CRISPR/Cas9核糖核蛋白复合物显微注射到受精卵中后，我们使用限制性片段长度的变化、桑格测序和高通量Illumina测序来检测靶基因中的诱导突变。我们观察到突变在类似的50%的个人筛选，和野生型和各种突变基因拷贝在这些人的比例表明，突变诱导注射后持续至少几个细胞周期。虽然A.在千孔藻中，指导RNA的适当设计使我们能够在多于一个亲本中同时诱导突变。因为A.尽管千孔珊瑚幼虫可以在实验室中被诱导定居并开始菌落形成，但基于CRISPR/Cas9的基因编辑应该可以在幼虫和成年珊瑚中进行严格的基因功能测试。
Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora. We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in similar to 50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.