Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation
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Evaluation Summary:
This study by Krug et al. uses the turquoise killifish, an emerging model for biomedical research, to generate a valuable live-imaging platform. Initially, the authors generate a transparent killifish they named Klara. Specifically, using optimized CRISPR approaches, they simultaneously inactivate three genes that are required for the formation of primary pigment cells in fish (melanophores, iridophores, xanthophores) and next, to monitor cell-cycle arrest and cellular senescence, they generate a cdkn1a-GFP reporter line using HDR-mediated integration. The paper would benefit from a further description of the HDR approach, the genetic models, and improved figures. Together, this platform will be an extremely valuable resource with broad application, including for aging research, physiology, toxicology, and regeneration.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Body pigmentation is a limitation for in vivo imaging and thus for the performance of longitudinal studies in biomedicine. A possibility to circumvent this obstacle is the employment of pigmentation mutants, which are used in fish species like zebrafish and medaka. To address the basis of aging, the short-lived African killifish Nothobranchius furzeri has recently been established as a model organism. Despite its short lifespan, N. furzeri shows typical signs of mammalian aging including telomere shortening, accumulation of senescent cells, and loss of regenerative capacity. Here, we report the generation of a transparent N. furzeri line by the simultaneous inactivation of three key loci responsible for pigmentation. We demonstrate that this stable line, named klara , can serve as a tool for different applications including behavioral experiments and the establishment of a senescence reporter by integration of a fluorophore into the cdkn1a (p21 ) locus and in vivo microscopy of the resulting line.
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Author Response
Reviewer #3 (Public Review):
The only substantial point I raise relates to the sexual selection (mate choice) part of the work. While it has no major effect on the overall conclusion, I think their interpretation needs to be reconsidered.
When reporting the results of mate choice experiment (L219ff), the authors state that males of wild and Klara type preferred wild-type females, because 75% of laid eggs belonged to wild-type females. However, another possibility is that Klara females had reduced fecundity, and the lower share of eggs had nothing to do with mate choice. In the same way, "90% of eggs were fertilized by wild-type males" (L223) is used to conclude that they were preferred by females (active mate choice). However, male success in N. furzeri is largely driven by male dominance (and not female mate …
Author Response
Reviewer #3 (Public Review):
The only substantial point I raise relates to the sexual selection (mate choice) part of the work. While it has no major effect on the overall conclusion, I think their interpretation needs to be reconsidered.
When reporting the results of mate choice experiment (L219ff), the authors state that males of wild and Klara type preferred wild-type females, because 75% of laid eggs belonged to wild-type females. However, another possibility is that Klara females had reduced fecundity, and the lower share of eggs had nothing to do with mate choice. In the same way, "90% of eggs were fertilized by wild-type males" (L223) is used to conclude that they were preferred by females (active mate choice). However, male success in N. furzeri is largely driven by male dominance (and not female mate choice) and it is more likely (and more precise to state) that wild-type males were more successful in male-male competition for access to females (and fertilize their eggs). This is especially so because wild-type males were larger (L. 322) and body size plays a major role in establishing dominance between N. furzeri males. This is then also pertaining to interpretation in discussion (L 318).
Concerning fecundity, we analyzed quantity and quality of eggs obtained from either klara or wild type breeding groups. As shown in Figure 3A we did not observe differences between klara and wild type fish. Thus, we conclude that fecundity is not reduced in klara females. Regarding males, we did not observe a size difference between the klara and wild type animals in this experiment (Fig. 3C), however, weight was different. As noted by the reviewer, this might influence male dominance and breeding success. We have been more explicit on this in the discussion of the revised version.
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Evaluation Summary:
This study by Krug et al. uses the turquoise killifish, an emerging model for biomedical research, to generate a valuable live-imaging platform. Initially, the authors generate a transparent killifish they named Klara. Specifically, using optimized CRISPR approaches, they simultaneously inactivate three genes that are required for the formation of primary pigment cells in fish (melanophores, iridophores, xanthophores) and next, to monitor cell-cycle arrest and cellular senescence, they generate a cdkn1a-GFP reporter line using HDR-mediated integration. The paper would benefit from a further description of the HDR approach, the genetic models, and improved figures. Together, this platform will be an extremely valuable resource with broad application, including for aging research, physiology, toxicology, and …
Evaluation Summary:
This study by Krug et al. uses the turquoise killifish, an emerging model for biomedical research, to generate a valuable live-imaging platform. Initially, the authors generate a transparent killifish they named Klara. Specifically, using optimized CRISPR approaches, they simultaneously inactivate three genes that are required for the formation of primary pigment cells in fish (melanophores, iridophores, xanthophores) and next, to monitor cell-cycle arrest and cellular senescence, they generate a cdkn1a-GFP reporter line using HDR-mediated integration. The paper would benefit from a further description of the HDR approach, the genetic models, and improved figures. Together, this platform will be an extremely valuable resource with broad application, including for aging research, physiology, toxicology, and regeneration.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In their manuscript, Krug et al describe a CRISPR/Cas9 knock-out strategy for the creation of a pigment-less killifish (Nothobranchius furzeri) they term "klara". They target and inactivate three genes in parallel (mitfa, ltk, csf1ra). They employ the generated mutant offspring for studying mating preference as well as additional genome editing (knock-out of slc452) or the HDR-mediated knock-in to generate an inducible model for aging (inducible NTR mediated cell death by tagging the ORF of cdkn1a).
The authors present a valuable resource, a large bouquet of different, well-designed and described controlled experiments. The authors demonstrate the versatility of the established tool that may be of immediate use for the Noto community. Given the efficiency of the triple inactivation, the de novo inactivation …
Reviewer #1 (Public Review):
In their manuscript, Krug et al describe a CRISPR/Cas9 knock-out strategy for the creation of a pigment-less killifish (Nothobranchius furzeri) they term "klara". They target and inactivate three genes in parallel (mitfa, ltk, csf1ra). They employ the generated mutant offspring for studying mating preference as well as additional genome editing (knock-out of slc452) or the HDR-mediated knock-in to generate an inducible model for aging (inducible NTR mediated cell death by tagging the ORF of cdkn1a).
The authors present a valuable resource, a large bouquet of different, well-designed and described controlled experiments. The authors demonstrate the versatility of the established tool that may be of immediate use for the Noto community. Given the efficiency of the triple inactivation, the de novo inactivation may be more time and cost-efficient compared to the traditional sharing of the mutant animals.
When creating transgenic lines via HDR-mediated integration of donor sequences, the authors use the advanced protection of the donor construct by the addition of a 5'biotin. They validate integration by PCR genotyping and sequencing. However, these "proper" PCR bands can derive from in vitro recombination during the PCR (Won and Dawid, PlosOne2017), if a large number of cycles are used during PCR amplification. While PCR is often misleading, Southern Blot analysis delivers robust and unambiguous results. Here a single-copy integration is not relevant to the message of the manuscript.
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Reviewer #2 (Public Review):
Krug et. al. have generated a transparent African killifish line, klara, for in vivo imaging to address fundamental questions regarding aging, regeneration, and others. This line was generated by simultaneously targeting three key loci (mitfa, Itk, and csf1ra) responsible for the formation of pigmentation using CRISPR/Cas9-mediated gene knockout. Further, the authors established a potential senescence reporter line in the background of the klara line, suggesting that additional genetic manipulations can be done in this special genetic background. Such a feature enhances the application of this tool in different biological contexts of interest. Although similar crystal-clear fish is already present in zebrafish, such an important tool is not present in the attractive aging and regeneration model, N. furzeri. …
Reviewer #2 (Public Review):
Krug et. al. have generated a transparent African killifish line, klara, for in vivo imaging to address fundamental questions regarding aging, regeneration, and others. This line was generated by simultaneously targeting three key loci (mitfa, Itk, and csf1ra) responsible for the formation of pigmentation using CRISPR/Cas9-mediated gene knockout. Further, the authors established a potential senescence reporter line in the background of the klara line, suggesting that additional genetic manipulations can be done in this special genetic background. Such a feature enhances the application of this tool in different biological contexts of interest. Although similar crystal-clear fish is already present in zebrafish, such an important tool is not present in the attractive aging and regeneration model, N. furzeri. This useful platform generated by the authors will facilitate the application of in vivo imaging to dissect the molecular basis of aging and regeneration. However, insufficient characterization and application of the klara line and cdkn1a (p21)-GFP line have been done in this manuscript.
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Reviewer #3 (Public Review):
Krug et al. used emerging model species in biomedical research, Nothobranchius furzeri, to construct a triple mutant line that lacks all three major pigments found in fish (melanophores, iridophores, xanthophores). It demonstrates clearly that multiple genes can be inactivated simultaneously in this species, and that a new line can be a source of additional genetic manipulations. This is because their condition, vigour, and fecundity are standard compared to the wild type, which is convincingly demonstrated.
The introduction is appropriate and results generally correctly report what has been achieved, which is then adequately addressed in the discussion. Methods, as far as I can estimate, are sufficient to replicate the work.
The only substantial point I raise relates to the sexual selection (mate choice) …
Reviewer #3 (Public Review):
Krug et al. used emerging model species in biomedical research, Nothobranchius furzeri, to construct a triple mutant line that lacks all three major pigments found in fish (melanophores, iridophores, xanthophores). It demonstrates clearly that multiple genes can be inactivated simultaneously in this species, and that a new line can be a source of additional genetic manipulations. This is because their condition, vigour, and fecundity are standard compared to the wild type, which is convincingly demonstrated.
The introduction is appropriate and results generally correctly report what has been achieved, which is then adequately addressed in the discussion. Methods, as far as I can estimate, are sufficient to replicate the work.
The only substantial point I raise relates to the sexual selection (mate choice) part of the work. While it has no major effect on the overall conclusion, I think their interpretation needs to be reconsidered.
When reporting the results of mate choice experiment (L219ff), the authors state that males of wild and Klara type preferred wild-type females, because 75% of laid eggs belonged to wild-type females. However, another possibility is that Klara females had reduced fecundity, and the lower share of eggs had nothing to do with mate choice. In the same way, "90% of eggs were fertilized by wild-type males" (L223) is used to conclude that they were preferred by females (active mate choice). However, male success in N. furzeri is largely driven by male dominance (and not female mate choice) and it is more likely (and more precise to state) that wild-type males were more successful in male-male competition for access to females (and fertilize their eggs). This is especially so because wild-type males were larger (L. 322) and body size plays a major role in establishing dominance between N. furzeri males. This is then also pertaining to interpretation in discussion (L 318).
While I think this needs to be corrected to avoid misinterpretation, it has a minor impact on the overall high standard of the work or on general interpretation.
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