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

Stundl, Jan and Soukup, Vladimir and Franěk, Roman and Pospisilova, Anna and Psutkova, Viktorie and Pšenička, Martin and Cerny, Robert and Bronner, Marianne E. and Medeiros, Daniel Meulemans and Jandzik, David (2022) Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates. Frontiers in Cell and Developmental Biology, 10 . Art. No. 750833. ISSN 2296-634X. PMCID PMC8867083. doi:10.3389/fcell.2022.750833. https://resolver.caltech.edu/CaltechAUTHORS:20220322-742455000

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.3389/fcell.2022.750833DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc8867083/PubMed CentralArticle
ORCID:
AuthorORCID
Stundl, Jan0000-0002-3740-3378
Soukup, Vladimir0000-0002-1914-283X
Franěk, Roman0000-0002-3464-1872
Pospisilova, Anna0000-0002-8252-0709
Pšenička, Martin0000-0002-3808-7856
Cerny, Robert0000-0002-0022-0199
Bronner, Marianne E.0000-0003-4274-1862
Medeiros, Daniel Meulemans0000-0002-8182-6028
Jandzik, David0000-0002-4239-0014
Additional Information:© 2022 Stundl, Soukup, Franěk, Pospisilova, Psutkova, Pšenička, Cerny, Bronner, Medeiros and Jandzik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 31 July 2021; Accepted: 17 January 2022; Published: 10 February 2022. We thank Marek Rodina, David Gela, Michaela Fučíková, and Martin Kahanec for their essential help with sterlet spawns; Vojtěch Miller and Štěpánka Novotná for technical assistance; Radek Šanda for allowing us to use the stereoscopic microscope with Z-stacking. Computational resources were supplied by the Ministry of Education, Youth and Sports of the Czech Republic under the Projects CESNET (Project No. LM2015042) and CERIT-Scientific Cloud (Project No. LM2015085) provided within the program Projects of Large Research, Development and Innovations Infrastructures. D.J. thanks Tyler Square for numerous discussions and priceless advice on everything CRISPR-related and Brent Hawkins for late night fish conversations and insightful comments on the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2022.750833/full#supplementary-material This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 897949 (to JS). VS was supported by Charles University Research Centre program No. 204069, Charles University Grant SVV 260571/202, and the grant of the Czech Science Foundation GACR 18-04580S. The work of RF and MP was supported by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (LM2018099), Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and Czech Science Foundation (20-23836S). RC was supported by the Czech Science Foundation GACR 19-18634S. MB was supported by National Institutes of Health grant R35NS111564. DM was supported by National Institutes of Health grant NIDCR R21 RDE025940A and National Science Foundation grant IOS 1656843. DJ was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 751066 and by the Scientific Grant Agency of the Slovak Republic VEGA grant no. 1/0450/21. Author Contributions. DJ conceived and designed the project, JS, VS, RF, AP, VP, MP, and DJ performed the experiments, JS and DJ collected and interpreted the data, DJ, DM, and JS wrote the manuscript. All authors discussed the results and contributed to the final manuscript. Data Availability Statement. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. The animal study was reviewed and approved by The Animal Research Committee of 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 Ministry of Agriculture of the Czech Republic (MSMT-12550/2016-3). Author Contributions. DJ conceived and designed the project, JS, VS, RF, AP, VP, MP, and DJ performed the experiments, JS and DJ collected and interpreted the data, DJ, DM, and JS wrote the manuscript. All authors discussed the results and contributed to the final manuscript.
Funders:
Funding AgencyGrant Number
Marie Curie Fellowship897949
Charles University204069
Charles UniversitySVV 260571/202
Czech Science FoundationGACR 18-04580S
Ministry of Education, Youth and Sports (Czech Republic)LM2018099
Ministry of Education, Youth and Sports (Czech Republic)CZ.02.1.01/0.0/0.0/16_025/0007370
Czech Science Foundation20-23836S
Czech Science FoundationGACR 19-18634S
NIHR35NS111564
NIHR21 RDE025940A
NSFIOS-1656843
Marie Curie Fellowship751066
Scientific Grant Agency (Slovakia)1/0450/21
Ministry of Education, Youth and Sports (Czech Republic)LM2015042
Ministry of Education, Youth and Sports (Czech Republic)LM2015085
Subject Keywords:CRISPR/Cas9, targeted mutagenesis, non-teleost fish, sturgeon, vertebrates, development, evolution, evo-devo
PubMed Central ID:PMC8867083
DOI:10.3389/fcell.2022.750833
Record Number:CaltechAUTHORS:20220322-742455000
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220322-742455000
Official Citation:Stundl J, Soukup V, Franěk R, Pospisilova A, Psutkova V, Pšenička M, Cerny R, Bronner ME, Medeiros DM and Jandzik D (2022) Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates. Front. Cell Dev. Biol. 10:750833. doi: 10.3389/fcell.2022.750833
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114009
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Mar 2022 14:54
Last Modified:23 Mar 2022 14:54

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