Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor
Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor
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DOI：
10.1073/pnas.1920779117
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发表时间：
2020-11-17
影响因子：
11.1
通讯作者：
Pringle, John R.
中科院分区：
文献类型：
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作者：
Cleves, Phillip A.;Tinoco, Amanda I.;Pringle, John R.
造礁珊瑚是受到包括气候变化在内的人为压力威胁的关键物种。为了研究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，产生了许多关于特定基因和分子途径作用的假设。然而，由于缺乏珊瑚或密切相关的刺胞动物的遗传工具，通常不可能严格测试这些假设。CRISPR技术似乎可以缓解这个问题。事实上，我们在这里表明，将单向导RNA/Cas9核糖核蛋白复合物显微注射到珊瑚Acropora millepora的受精卵中可以产生足够高的突变频率，以检测注射一代中的明确表型。部分基于海葵模型系统中的实验，我们靶向编码热休克转录因子1（HSF 1）的基因，并获得了>90%的基因拷贝突变的幼虫。突变体幼虫在27摄氏度下存活良好，但在34摄氏度下迅速死亡，在野生型（WT）幼虫或单独注射Cas9的幼虫的实验期间，该温度没有产生可检测的死亡率。我们的结论是，HSF 1功能（推测其诱导的基因在热应激反应）在珊瑚中起着重要的保护作用。更广泛地说，我们的结论是，珊瑚中的CRISPR诱变应该允许对幼虫和成年珊瑚中的基因功能进行广泛而严格的测试。
Reef-building corals are keystone species that are threatened by anthropogenic stresses including climate change. To investigate corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating many 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 or closely related cnidarians. CRISPR technology seems likely to alleviate this problem. Indeed, we show here that microinjection of single-guide RNA/Cas9 ribonucleoprotein complexes into fertilized eggs of the coral Acropora millepora can produce a sufficiently high frequency of mutations to detect a clear phenotype in the injected generation. Based in part on experiments in a sea-anemone model system, we targeted the gene encoding Heat Shock Transcription Factor 1 (HSF1) and obtained larvae in which >90% of the gene copies were mutant. The mutant larvae survived well at 27 degrees C but died rapidly at 34 degrees C, a temperature that did not produce detectable mortality over the duration of the experiment in wild-type (WT) larvae or larvae injected with Cas9 alone. We conclude that HSF1 function (presumably its induction of genes in response to heat stress) plays an important protective role in corals. More broadly, we conclude that CRISPR mutagenesis in corals should allow wide-ranging and rigorous tests of gene function in both larval and adult corals.