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

Abstract

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 We targeted the genes encoding fibroblast growth factor 1a (), green fluorescent protein (), and red fluorescent protein (). 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 , appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because 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.

Evidence for a role of protein phosphorylation in the maintenance of the cnidarian–algal symbiosis

“…Additionally, the establishment of an Aiptasia or coral endodermal cell line would further allow characterization of protein phosphorylation by means of chemical inhibition of kinases or phosphatases that specifically phospho‐/dephosphorylate proteins of interest. Ultimately, functional validation of phosphorylation by phosphosite‐directed mutagenesis thanks to emerging genome editing techniques in the field (Cleves, Strader, Bay, Pringle, & Matz, ) could unveil regulatory phosphosites.…”

Tropicalisation of temperate reefs: Implications for ecosystem functions and management actions

“…Conceptually, the idea includes one or both of (a) enhancing genetic adaptation (resilience) of a species to current or future environmental stressors such as climate change and (b) assisted (enhanced) gene flow (Aitken & Whitlock, ) by which superior genes or genotypes are introduced and propagated through the target population. For the former, possibilities range from selective breeding of more resilient individuals to the creation of genetically modified organisms via direct genetic manipulations (van Oppen et al, ), which has recently been piloted using CRISPR/Cas9‐genome editing (Cleves, Strader, Bay, Pringle, & Matz, ). For the later, transplanting adapted individuals (assisted migration) or “seeding” challenged populations with propagules from enhanced individuals have both been contemplated.…”

Considerations for maximizing the adaptive potential of restored coral populations in the western Atlantic

“…, Cleves et al. ). Challenges include our rudimentary understanding of the genetic basis of traits that will be essential in the future and limited knowledge of their interactions (i.e., trade‐offs; Muller et al.…”