CRISPR/Cas9-mediated genome editing in a reef-building coral

Abstract

<p id="d1857444e225">Coral reefs are biodiversity hotspots of great ecological, economic, and aesthetic importance. Their global decline due to climate change and other anthropogenic stressors has increased the urgency to understand the molecular bases of various aspects of coral biology, including the interactions with algal symbionts and responses to stress. Recent genomic and transcriptomic studies have yielded many hypotheses about genes that may be important in such processes, but rigorous testing of these hypotheses will require the generation of mutations affecting these genes. Here, we demonstrate the efficient production of mutations in three target genes using the recently developed CRISPR/Cas9 gene-editing technique. By clarifying aspects of basic coral biology, such genetic approaches should also provide a more solid foundation for coral-conservation efforts. </p><p class="first" id="d1857444e228">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 <i>Acropora millepora</i>. We targeted the genes encoding fibroblast growth factor 1a ( <i>FGF1a</i>), green fluorescent protein ( <i>GFP</i>), and red fluorescent protein ( <i>RFP</i>). 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 ∼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 <i>A. millepora</i>, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because <i>A. millepora</i> 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. </p>