Rapid and precise genome engineering in a naturally short-lived vertebrate

  1. Claire N Bedbrook
  2. Ravi D Nath
  3. Rahul Nagvekar
  4. Karl Deisseroth
  5. Anne Brunet

  • Curated by eLife

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    **eLife assessment
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    Within this paper, the authors describe a rapid and easy-to-implement CRISPR/Cas9-mediated knock-in approach to precisely insert large transgenes in the African turquoise killifish. The established method will be instrumental for many researchers working with unusual model species, and, in particular, will expand the killifish community toolbox. It will revolutionize the field and bring the killifish, an emerging animal model in aging biology and disease modeling in vertebrates, into the spotlight even more.

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Abstract

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

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  1. Version published to 10.7554/elife.80639 on eLife
  2. eLife

    **eLife assessment
    **
    Within this paper, the authors describe a rapid and easy-to-implement CRISPR/Cas9-mediated knock-in approach to precisely insert large transgenes in the African turquoise killifish. The established method will be instrumental for many researchers working with unusual model species, and, in particular, will expand the killifish community toolbox. It will revolutionize the field and bring the killifish, an emerging animal model in aging biology and disease modeling in vertebrates, into the spotlight even more.

  3. eLife

    Reviewer #1 (Public Review):

    This manuscript reports on a rapid and precise CRISPR/Cas9-mediated knock-in approach in the African turquoise killifish, an emerging vertebrate animal and gerontology model. More specifically, it describes an easily adoptable method to efficiently insert fluorescent reporters of different sizes at various genomic loci and to drive cell-type- and tissue-specific expression. This methodology will allow the development of humanized disease models and of cell-type specific molecular probes to study complex vertebrate biology, including aging biology, in the killifish. While this knock-in methodology is already widely used in common vertebrate animal models, the efficient generation of stable lines with germline transmission has been missing in killifish. As killifish have the shortest generation time of vertebrate animal models in laboratory conditions, show a rapid sexual maturity, and a short lifespan, the established method enables the generation of stable lines of homozygous transgenic vertebrate animals in 2-3 months. Overall, we believe this first report on efficient long (1.8kb) construct knock-in using CRISPR/Cas9 in the killifish establishes the killifish as a system for precise genetic engineering at scale, which has been challenging so far in vertebrates.

    The establishment of this methodology will have a major impact in the field and be of extreme use within the scientific community. It will allow the development of scalable human disease models and integrate both genetics and age as risk factors, thus having the potential to identify future therapeutic targets for age-related diseases. It also has a generic character as the generated protocol can serve as a template for knock-in approaches in other emerging model organisms.

    Although the reported data are of major interest and relevance to the scientific field, they are, as yet not sufficiently shown in convincing figures. The methodology is state-of-the-art and entails an extensive set of molecular, biochemical, and morphological/imaging technologies. While most of the data are nicely presented and accompanied by illustrative figures, the manuscript would benefit from the inclusion of a more detailed material and methods section, and a little more elaboration on morphometrical expression data in the results section, e.g, expression shown for all the studied genes in the larval fish, and a more critical discussion, that also highlights a few of the limitations, e.g., those related to the fast generation of homozygous F1 fish.

  4. eLife

    Reviewer #2 (Public Review):

    The manuscript "Rapid and precise genome engineering in a naturally short-lived vertebrate" describes the development of a CRISPR- based knock-in technology in Nothobranchius furzeri, or the African turquoise killifish, an innovative model species for studying aging and age-related disorders. While Tol2 systems had been demonstrated to be successful in generating reporter killifish lines, endogenous reporters via knock-in had not been reported so far. The major strength of the paper is that the authors show that they have been successful in developing 5 different knock-in fish lines with large inserts (up to 1.8kb) with high efficiency. They have inserted single or dual fluorescent reporters and demonstrated expression in line with the expected pattern. This is a breakthrough in the field and this method can be instrumental for many researchers working with unusual model species, and in particular, will expand the killifish community toolbox.

    While this is very promising, the paper would benefit from a more rigorous validation of the KI lines that were generated. The authors did not show a co-localisation of the target gene expression with the reporter to prove bona fide reporting. In addition, it was not clear whether the KI affects the endogenous expression level of the target genes. The targeting efficiency of the method is high, but the quantifications are based on rather limited numbers of animals, which might not yet be very robust. A larger number of animals would have strengthened the efficiency conclusion.

    The figures of the manuscript are well designed and support the conclusions, but several contain information that is not discussed in the main text, such as (un)expected bands on gels, reporter staining in WT animals, and unusual staining patterns. The body text seems to ignore these and only discusses findings that are in line with the story. A key point to the efficiency of the method seems to be a chemical modification of the repair template, which was not disclosed in the method section which at the moment hampers replication.

    Finally, the discussion is brief and does not benchmark the method to other CRISPR-based KI methods in Xenopus or more typical model species such as mouse.

    In conclusion, this paper describes a breakthrough method for a rising animal model that would benefit from a more thorough validation. Full disclosure of the methodology will boost the generation of genetically edited killifish lines and aid in the establishment of this promising animal model.

  5. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors detail an exciting protocol to knock-in c-terminal tags at the endogenous locus of a gene of interest in the short-lived vertebrate, the African Turquoise Killifish. The technique is clearly explained and will be a significant advancement for the field. The method relies on the injection of a cocktail containing cas9 protein, proprietary-modified gRNA and dsDNA ordered from IDT, and a chemical enhancer of HDR.

    I believe the authors demonstrate that killifish is a tractable emerging system to integrate stable fluorescent tags at desired loci. The method should be easy to reproduce since the components are all commercially available, but the proprietary nature of the modifications could make it a pricey technique for smaller labs. A protospacer adjacent motif (PAM) sequence near the desired insertion site is still a restriction using this method. That being said, I think this represents a significant advancement in knock-in methods that could be adopted in other systems. This manuscript is rather simple and straightforward, and I do not have additional criticisms or critiques.

    As a side note, I think the brain sections look very professional, but since I am not a neurobiologist I will defer to the other reviewers about the accuracy and claims about the regions labeled.

    For additional context, I suggest reading Wierson et al. 2020 and Seleit et al. 2022, which can both be found in the reference section.

  6. Version published to 10.1101/2022.05.25.493454v1 on bioRxiv