Establishment of knockout adult sea urchins by using a CRISPR‐Cas9 system

Abstract

Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc‐finger nuclease (ZFN) and transcription activator‐like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)‐CRISPR‐associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high‐efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA‐injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off‐target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR‐Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.

The biosynthetic diversity of the animal world

“…Coloration in the sea urchin is due to an aromatic polyketide-like product, echinochrome, so it was speculated that Pks1 might be the urchin echinochrome synthase. Recently, a stable knockout was created that showed an absolute loss of pigment through the life cycle, further supporting this assignment (32). Pks1 was representative of a larger family of animal PKSs evidenced in genomes and transcriptomes (33).…”

Regulation of dynamic pigment cell states at single-cell resolution

“…From an intense red sea star ( Fromia milleporella ) to a black and white striped brittle star ( Ophiactis savignyi ) to the variegated sea urchin ( Lytechinus variegatus), pigment is a pervasive feature of this phylum. Because of the molecular techniques now available for echinoderms, mechanisms controlling pigmentation in these animals are being revealed ( Hira et al, 2020 ; Liu et al, 2019 ; Wessel et al, 2020 ; Yaguchi et al, 2020 ). The purple sea urchin, Strongylocentrotus purpuratus , provides a molecularly tractable model organism to dissect the developmental importance of pigmented cells, and the biosynthesis of their pigment.…”

Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition

“…The enzymatic pathway to make pigment in sea urchins involves a large, iterative-type polyketide synthase expressed in the larvae exclusively in the pigment cells 14 . PKS-null (Cas9-gRNA mediated genomic mutation) or knockdown (morpholino antisense oligonucleotide; MASO) larvae in two different species (Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus) result in normal appearing development, but lacking in pigment [14][15][16] . A flavin-dependent monooxygenases (FMO3) also appears essential for larval pigmentation in S. purpuratus.…”