Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchinLytechinus pictus

Abstract

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

Induciblein vivogenome editing in the sea starPatiria miniata

“…Previous studies used CRISPR targeted genome editing in echinoderms to alter either structural (Lin et al, 2019; Mellott et al, 2017; Wessel et al, 2020) or regulatory (Pieplow et al, 2021) genes, and to create the first stable transgenic sea urchin lines (Liu et al, 2019; Vyas et al, 2022; Yaguchi and Yaguchi, 2022). However, these were not tunable for in vivo spatio-temporal regulation.…”

CRISPR/Cas9 knockin methodology for the sea urchin embryo

“…CRISPR technology has revolutionized the biological research world, making animals heretofore recalcitrant to genetic manipulation, accessible to analysis of specific gene functions. Building upon the demonstration of targeted gene mutations in the sea urchin (CRISPR knockout) (Fleming et al, 2021; Lin et al, 2019; Lin & Su, 2016; Liu et al, 2019; Vyas et al, 2022), investigators may now be able to insert exogenous DNA into specific locations in the genome (CRISPR knockin). Such Cas9‐mediated knockins will reveal sites of gene expression, and function.…”

“…CRISPR/Cas9 knockin will enable researchers to follow the expression of their favorite genes in live cells and embryos, even over multiple generations (Vyas et al, 2022) if the insertion is transmitted to the germline. Fundamental processes such as the biology of the pigment cells (using Sp PKS1 gene) and the biology of the germ cells (using Sp Nanos2; Oulhen & Wessel, 2016b), can now be explored in live embryos with this method.…”

Developmental staging and future research directions of the model marine tubewormHydroides elegans

“…There has been recent success in the genetic manipulation of related Annelids such as Platynereis dumerilii 51,52 and Capitella teleta 53,54 and we are optimistic that Hydroides will be amenable to genetic manipulation. Furthermore, improvements in husbandry and longterm culture will further facilitate generation of animal lines in Hydroides, enabling Hydroides to keep pace with advancements, like CRISPR gene editing, in other marine invertebrate larvae [55][56][57][58][59][60] .…”