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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 20; pp. 5235 - 5240
• Main Authors: Cleves, Phillip A., Strader, Marie E., Bay, Line K., Pringle, John R., Matz, Mikhail V.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.05.2018
• Series: From the Cover
• Subjects: Animals; Anthropogenic factors; Base Sequence; Biological Sciences; Climate change; Coral Reefs; Corals; CRISPR; CRISPR-Cas Systems; Eggs; Fibroblast Growth Factor 1 - antagonists & inhibitors; Fibroblast Growth Factor 1 - genetics; Fibroblast growth factors; Fluorescence; Gene Editing; Gene sequencing; Genes; Genetic modification; Genome; Genomes; Genomics; Green fluorescent protein; Green Fluorescent Proteins - antagonists & inhibitors; Green Fluorescent Proteins - genetics; Growth factors; Hypotheses; Larvae; Luminescent Proteins - antagonists & inhibitors; Luminescent Proteins - genetics; Mutation; Phenotype; Proteins; Red Fluorescent Protein; Ribonucleic acid; RNA; Sequence Homology; Threatened species; genome editing; CRISPR/Cas9; coral; Acropora millepora
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1722151115

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 ∼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.