Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates

Abstract

In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or “basal” vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants’ various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.

Identification of multiple transcription factor genes potentially involved in the development of electrosensory versus mechanosensory lateral line organs

“…The copyright holder for this preprint this version posted April 14, 2023. ; https://doi.org/10.1101/2023.04.14.536701 doi: bioRxiv preprint mechanosensory organs. These are good candidates for functional experiments using CRISPR/Cas9-mediated mutagenesis in sterlet (e.g., Chen et al, 2018;Baloch et al, 2019;Stundl et al, 2022), the next step to further our understanding of the development of these sensory (sister) cell types. Mississippi paddlefish (Polyodon spathula) embryos were purchased from Osage Catfisheries Inc. (Osage Beach, MO, USA) and reared at approximately 22°C in tanks with filtered and recirculating water (pH 7.2 ± 0.7, salinity of 1.0 ± 0.2 ppt).…”

“…The copyright holder for this preprint this version posted April 14, 2023. ; https://doi.org/10. 1101 In recent years, another chondrostean fish, the sterlet (a sturgeon, Acipenser ruthenus), has been developed as an experimentally tractable non-teleost model (see e.g., Saito and Psenicka, 2015;Chen et al, 2018;Baloch et al, 2019;Stundl et al, 2020;Du et al, 2020;Stundl et al, 2022). In contrast to the limited Mississippi paddlefish spawning season, many hundreds of sterlet embryos are available each week for up to several months in fully equipped laboratory research facilities.…”

Atoh1 is required for the formation of lateral line electroreceptors and hair cells, whereas Foxg1 represses an electrosensory fate

“…and M.P. ; Chen et al, 2018;Baloch et al, 2019;Stundl et al, 2022. ) We targeted the melanin-producing enzyme tyrosinase (Tyr) as a positive control, using eight different single-guide (sg) RNAs.…”

“…Fertilized sterlet (Acipenser ruthenus) eggs were obtained from the breeding facility at the Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czech Republic, and staged according to Dettlaff et al (1993). For detailed information about sterlet husbandry, in vitro fertilization and the rearing of embryos and yolk-sac larvae, see Stundl et al (2022). Upon reaching the desired developmental stages, embryos and yolk-sac larvae were euthanized by overdose of MS-222 (Sigma-Aldrich) and fixed in modified Carnoy's fixative (6 volumes 100% ethanol: 3 volumes 37% formaldehyde: 1 volume glacial acetic acid) for 3 hours at room temperature, dehydrated stepwise into 100% ethanol and stored at -20°C.…”

Pre-mandibular pharyngeal pouches in early non-teleost fish embryos

“…Finally, Sonic hedgehog (Shh), a potent pleiotropic morphogen, is differentially expressed in the embryonic pharyngeal endoderm and plays multiple roles throughout the development of the pharynx including the repression of Fgf signalling [58]. Mutation of Shh in the sturgeon sterlet resulted in severe pharyngeal phenotypes [59].…”