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  1. Author Response

    Reviewer #2 (Public Review):

    This manuscript describes a method for generating floxed conditional alleles in the zebrafish. The method employs the authors' previously reported GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to introduce a "UFlip" cassette that allows target genes to be "turned off" or "turned on" in a tissue-specific manner with appropriate cre driver lines. The authors provide data to show the efficacy of their new method by targeting hdac1, rbbp4, and rb1. Although a variety of other methods have recently been reported for gene inactivation in the zebrafish (many of which were cited and discussed by the authors of this manuscript), the authors method could provide some notable and significant advances including speed and ease of generation of conditional alleles and flexibility to generate "on" and "off" alleles at the same genomic locations. However, the authors would need to do more to provide additional and more quantitative data validating some of the important features and advantages of their method. A few of the most significant issues that should be addressed include:

    Better assessment of the efficiency of cre/dre "flipping" of integrated constructs.

    It's important that the authors provide data showing not just that inversion of their integrated constructs can happen, but quantitatively measuring how efficiently this occurs. The authors should provide qPCR or other measurements to assess % inversion of their constructs via injected and transgene-driven cre. The authors should also provide data quantitating the % efficiency of dre/rox inversion used to turn "off" alleles into "on" alleles and vice-versa (see Supp Fig. 5).

    We performed genomic qPCR to assess efficiency of Cre-mediated inversion. Cre injection led to robust inversion efficiency of 82-93%. As expected, the efficiency of inversion with transgenic ascl1b-2A-Cre and neurod1-2A-Cre was lower, ranging from 20-30%, due to the expression of these Cre drivers in a subset of cells in the embryo.

    The utility of the rox sites is illustrated in Figure 3. We injected Dre mRNA injection into rbbp4off/+ embryos and screened adults for germline transmission of an inverted rbbp4off to rbbp4on allele (Figure 3). This was highly efficient, >50% of adults that were injected with Dre mRNA as embryos transmitted the inverted allele to progeny, with a frequency of 7-43% inheriting the rbbp4on allele. Starting with a UFlip-2A-mRFP “off” allele, which we recovered at frequencies of 12.5% (rbbp4off is61) and 14% (rb1off is63) (Table 2), it is easy to recover the conditional “on” allele by Dre-mediated inversion.

    Although the authors provide junction qRT-PCR suggesting efficient transcript blocking by "off" insertion alleles (eg Fig 2B,J) it would also be useful to further validate and explore the effects of "off" UFlip transgene insertions on expression of targeted genes by similar qRT-PCR on upstream and downstream exons in control vs. het or homozygous transgene insertion animals to assess whether truncated transcripts are degrading, and whether expression of downstream exons is indeed absent.

    We used both RT-qPCR and phenotypic assessment to determine the impact of the cassette integration on gene expression and gene activity. As suggested, we included RT-qPCR results for the wildtype exon-exon splicing that would be disrupted by the cassette integration in the intron. We also examined splicing between exons downstream of the integration. These results demonstrate that in the gene “off” orientation endogenous gene expression is 99% knocked down. Integration in the passive “on” orientation did reduce expression in homozygotes (rbbp4on/on Figure 4 J reduced by 40.7%; rb1on/on Figure 7 J 1 reduced by 17.1%). However, the reduction in expression did not lead to mutant phenotype in heterozygotes ,or in combination with loss of function alleles (Figure 4 K-N; Figure 7, K-P), indicating gene activity was not disrupted. This may be gene dependent.

    Better validation that "null phenotypes" can be generated.

    Although the qRT-PCR data the authors provide suggests efficient transcript blocking by "off" insertion alleles, the authors need to strengthen some of their data showing that null phenotypes are being generated by these alleles. In many cases the authors provide only anecdotal images, describe relatively generic phenotypes, or provide quantitative measurements of mutant phenotypes (eg pH3 positive cells) that lack key positive controls such as comparable quantitative measurements on previously generated bona fide "null mutant" alleles of the same genes. All of this is important to demonstrate that this method can generate robust phenotypes that are both qualitatively and quantitatively comparable to null mutants.

    For each conditional allele that was isolated we performed RT-qPCR on embryos from each genotypic class of a conditional allele incross, to determine the impact of the targeted allele on gene expression (Figures 4, 5).

    Quantification of mutant phenotypes and statistical analyses to determine significance have been performed for 1. The characterization of the conditional alleles 2. The assessment of conditional rescue or inactivation. Data plots with p values are included in all figures and figure legends.

    Demonstrate tissue-specific "flipping on." One of the major points of novelty and most exciting features of the authors methods over other recently reported approaches is the potential to carry out tissue-specific gene activation (by cre-flipping on "UFlip-Off" alleles). This would be an exceptionally useful and powerful new tool for fish researchers (and others). Surprisingly, this particularly exciting feature is curiously unexplored in this manuscript, although the authors do generate a number of UFlip-On alleles. The impact and significance of this manuscript would be substantially increased by a well-validated and quantitated demonstration of tissue-specific activation of a "UFlip Off" allele, perhaps demonstrating rescue and lack of rescue of tissue-specific specific mutant phenotypes by activation using different cre drivers.

    Thank you for this suggestion. We performed the following experiments:

    • Tissue-specific conditional rescue: Using the conditional rbbp4off and rb1off alleles we demonstrate the ability to turn a gene “on” and rescue a loss of function phenotype. Conditional rescue of rbbp4off to “on” with ascl1b-2A-Cre lead to a reduction in apoptosis in the midbrain, although not significant (Figure 5). Conditional rescue with neurod1-2A-Cre did not lead to any apparent rescue, suggesting rbbp4 isn’t required in this cell population for survival.

    Conditional rescue of rb1off to on with neurod1-2A-Cre was clear and robust (Figure 8 O), suppressing the mutant phenotype with significant reduction in the number of mutant cells throughout the midbrain and retina. The same rescue was observed with the ascl1b-2A-Cre, but in the interest of space we did not include this data in Figure 8.

    • Tissue-specific conditional inactivation: Using the conditional rbbp4on and rb1on alleles, we demonstrate using cell type specific proneural Cre drivers that each gene is required in the progenitor population during brain development. Conditional inactivation of rbbp4on to “off” with ascl1b-2A-Cre leads to apoptosis in the developing midbrain optic tectum (Figure 6, Figure 6-figure supplement 1 and 2). Conditional inactivation of rb1on to “off” with neurod1-2A-Cre leads to inappropriate cell cycle entry in the midbrain tectum and retina. (Figure 9, Figure 9-figure supplement 1).

    Together with the conditional inactivation experiments using ascl1-2A-Cre and neurod1-2A-Cre that validate the cell type-specific requirement for these genes, the data demonstrating cell type-specific rescue is compelling.

    Future work using tamoxifen responsive CreERT2 drivers would help refine these analyses of cell type-specific requirements for gene function. We believe our current study provides a solid foundation demonstrating both conditional inactivation and rescue are possible.

    • Ubiquitous conditional inactivation and rescue: By Cre injection, we demonstrate robust conditional inactivation (“on” to “off”) and induction of mutant phenotypes for both genes (Figures 6 and 9). We also demonstrate robust conditional activation (“off” to “on”) and rescue of mutant phenotypes for both genes (Figures 5 and 8).

    • In all cases we provide rigorous quantification and statistical analysis of phenotypic changes and measure the frequency of Cre-mediated recombination.

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  2. Evaluation Summary:

    This technical paper describes a novel strategy for conditional mutagenesis in zebrafish. The method employs the authors' previously reported GeneWeld CRISPR/Cas9 targeted integration strategy to allow target genes to be "turned off" or "turned on" in a tissue-specific manner. Once fully validated, the approach would provide a valuable new addition to the "zebrafish genetic toolkit" that is likely to be widely used for assessing cell- and tissue-specific gene function in this model organism.

    (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. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    In the present technical report the authors develop a novel and simple strategy to generate and efficiently isolate cre/lox responsive alleles that in the right orientation are highly mutagenic. These allele offer in principle a powerful tool for the tissue specific analysis of gene function giving the ability to inactivate the gene of interest with high temporal and spatial control. The mutagenic targeting cassette contains a marker reporter for the efficient isolation of putative carriers following the integration in injected zebrafish embryos. The cassette in the mutagenic orientation also potentially labels the disrupted gene with a fluorescent tag, with the intent of following the single cells (and their full lineage) in which gene disruption has occurred. This approach is applied to three test loci comparing for which the same group had generated classical null alleles containing small deletions in the coding region.

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  4. Reviewer #2 (Public Review):

    This manuscript describes a method for generating floxed conditional alleles in the zebrafish. The method employs the authors' previously reported GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to introduce a "UFlip" cassette that allows target genes to be "turned off" or "turned on" in a tissue-specific manner with appropriate cre driver lines. The authors provide data to show the efficacy of their new method by targeting hdac1, rbbp4, and rb1. Although a variety of other methods have recently been reported for gene inactivation in the zebrafish (many of which were cited and discussed by the authors of this manuscript), the authors method could provide some notable and significant advances including speed and ease of generation of conditional alleles and flexibility to generate "on" and "off" alleles at the same genomic locations. However, the authors would need to do more to provide additional and more quantitative data validating some of the important features and advantages of their method. A few of the most significant issues that should be addressed include:

    Better assessment of the efficiency of cre/dre "flipping" of integrated constructs.

    It's important that the authors provide data showing not just that inversion of their integrated constructs can happen, but quantitatively measuring how efficiently this occurs. The authors should provide qPCR or other measurements to assess % inversion of their constructs via injected and transgene-driven cre. The authors should also provide data quantitating the % efficiency of dre/rox inversion used to turn "off" alleles into "on" alleles and vice-versa (see Supp Fig. 5).

    Although the authors provide junction qRT-PCR suggesting efficient transcript blocking by "off" insertion alleles (eg Fig 2B,J) it would also be useful to further validate and explore the effects of "off" UFlip transgene insertions on expression of targeted genes by similar qRT-PCR on upstream and downstream exons in control vs. het or homozygous transgene insertion animals to assess whether truncated transcripts are degrading, and whether expression of downstream exons is indeed absent.

    Better validation that "null phenotypes" can be generated.

    Although the qRT-PCR data the authors provide suggests efficient transcript blocking by "off" insertion alleles, the authors need to strengthen some of their data showing that null phenotypes are being generated by these alleles. In many cases the authors provide only anecdotal images, describe relatively generic phenotypes, or provide quantitative measurements of mutant phenotypes (eg pH3 positive cells) that lack key positive controls such as comparable quantitative measurements on previously generated bona fide "null mutant" alleles of the same genes. All of this is important to demonstrate that this method can generate robust phenotypes that are both qualitatively and quantitatively comparable to null mutants.

    Demonstrate tissue-specific "flipping on."

    One of the major points of novelty and most exciting features of the authors methods over other recently reported approaches is the potential to carry out tissue-specific gene activation (by cre-flipping on "UFlip-Off" alleles). This would be an exceptionally useful and powerful new tool for fish researchers (and others). Surprisingly, this particularly exciting feature is curiously unexplored in this manuscript, although the authors do generate a number of UFlip-On alleles. The impact and significance of this manuscript would be substantially increased by a well-validated and quantitated demonstration of tissue-specific activation of a "UFlip Off" allele, perhaps demonstrating rescue and lack of rescue of tissue-specific specific mutant phenotypes by activation using different cre drivers.

    Was this evaluation helpful?