ExTaSy: A swappable CRISPR platform for endogenous tagging in Drosophila melanogaster

  1. Sean Hubbert
  2. Zara Valliji
  3. Sadeen Abu-Hananah
  4. Sarah Phal
  5. Marina Luchner
  6. Emma Watts
  7. Giulia Biasi
  8. Korneel Hens
  9. Sebastian Kittelmann

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Abstract

Gene tagging enables functional analysis of proteins in vivo . Currently existing technologies in Drosophila suffer from drawbacks including limited flexibility, copy number variation, and/or imprecise expression. To address this, we have developed the Exchangeable Tagging System (ExTaSy), a CRISPR/Cas9-based platform which introduces a 3XHA tag into the endogenous gene locus. Importantly, the 3XHA can be subsequently exchanged for other tags using fly crosses, making the platform highly versatile and accessible. Simultaneously, an excisable transgenic marker allows virtually scarless locus modification. Here, we report successful tagging of 55 different loci, which shows that ExTaSy can be used to tag genes across the Drosophila genome and demonstrate its versatility for functional studies. We also developed a software that automates guide RNA and homology arm design, aiding efficient synthesis of transgenesis constructs. This novel technology will significantly improve our ability to visualize and manipulate genes using various applications in vivo while maintaining endogenous expression levels and genetic background.

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  1. Review Commons

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    Reply to the reviewers

    Response to the Reviewers

    We thank three anonymous Reviewers for their careful examination of our manuscript. Below, we provide a point-by-point response.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    1. EVIDENCE, REPRODUCIBILITY AND CLARITY Summary

    Hubbert and colleagues describe ExTaSy, a CRISPR-Cas9-based platform for the endogenous tagging of proteins in Drosophila melanogaster. The system combines several established molecular tools into a single-vector framework: homology-directed repair (HDR) for the insertion of a 3XHA tag at the endogenous locus, piggyBac transposase-mediated near-scarless removal of a transgenic selection marker, and φC31 integrase-mediated recombination-mediated cassette exchange (RMCE) for subsequent tag swapping. The authors demonstrate the system across a set of 65 genomic loci and provide a bioinformatic pipeline to automate guide RNA and homology arm design.

    Major Comments

    1. Validation of knock-in lines is inadequate and does not reflect current standards in the field. The authors state that correct insertions were confirmed using "two PCRs per inserted fragment done with primers binding to the 5' and 3' ends of the inserted DNA and corresponding gene-specific validation primers." This strategy is well known to produce false positives, as it cannot distinguish correctly targeted single-copy integrants from concatemeric insertions at the target locus (e.g. Skryabin et al., 2020). The current standard for validating CRISPR-mediated knock-ins requires PCR amplification using primers that anneal outside the homology arms and span the entire inserted cassette. These reactions must be performed under conditions that minimise the formation of PCR chimeras, specifically low cycle numbers and use of a high-processivity polymerase. The authors should either provide data from such experiments for their characterised lines, or clearly acknowledge this limitation and qualify their efficiency estimates accordingly (see related point 2 below).

    __Response: __We originally opted for using primers that span a fragment from the inserted DNA into the genomic locus for ease of amplification, which is currently standard in the field (e.g., Kanca et al. 2022). We usually run these PCRs in a heterozygous background (before homozygous stocks are established or because tagged lines remain balanced), and the unmodified locus preferentially amplifies in a whole-fragment PCR. However, we have recently started running whole-fragment PCRs and plan to repeat them for all loci and will report the results in a revised version of the manuscript. We are also revising the manuscript to reflect the necessity (or at least preference) to perform insert-spanning PCRs.

    Reported efficiency metrics do not adequately distinguish correctly targeted integrants from marker-positive flies.

    A related concern is that many of the efficiency parameters reported in the manuscript appear to be based solely on the detection of the marker cassette. The 63.1% overall success rate, for example, seemingly reflects the recovery of DsRed-positive flies rather than of sequence validated, single-copy, on-target integrants. These are fundamentally different quantities, with only the latter being of practical value for the users of the described technique. The authors should either provide data that properly accounts for correct integration, or more carefully define what each reported metric represents and explicitly acknowledge the limitations of using marker presence as a proxy for successful knock-in.

    __Response: __The reviewer is correct that the numbers we report are DsRed-positive flies. However, most have been confirmed with end-of-fragment/locus spanning PCRs, so are on-target (although not necessarily single-copy; see comment #1). While we cannot categorically exclude off-target insertions, we have not observed any cases where the DsRed segregates independently of the targeted chromosome, which at least makes off-target insertions on other chromosomes highly unlikely. We will clarify in the text that the 63.1 % success rate relates to DsRed marker expression and insertion site-spanning PCR and acknowledge the limitations as suggested by the reviewer.

    The characterisation of tag exchange requires expansion or more careful framing of its scope.

    The possibility of exchanging tags through fly crosses rather than repeated microinjections is, in the view of this reviewer, the most practically useful feature of ExTaSy and the aspect most likely to drive community adoption. It is therefore important that this feature is characterised with sufficient rigour to allow prospective users to assess its reliability. In the current manuscript, tag exchange has been demonstrated at only five loci using a single replacement tag (sfGFP). The dataset includes one outright failure (the Met C-terminus) and one instance of an unexpected 9 bp insertion at the recombination site, leaving the success rates and failure modes across a broader range of loci and tags uncharacterised. The authors should either expand the tag exchange experiments to cover a more representative set of conditions, or frame the current data explicitly as a proof of concept and limit their conclusions about the practical utility of tag exchange accordingly. In either case, the value of this work to the community would be substantially increased if a collection of donor lines carrying the most commonly used tags for different applications, as the authors themselves enumerate in the Discussion, were generated and deposited at a public stock centre such as the VDRC concurrent with publication. On this note, it is also worth flagging that at present the plasmids described in this study have not yet been deposited at Addgene or the European Plasmid Repository, and that fly lines are available only on request. For a methods paper aimed at community adoption, deposition of reagents in publicly accessible repositories at the time of publication is the expected standard.

    __Response: __We are in the process of increasing the number of fly stocks for which tags have been exchanged and will be able to provide a more rigorous characterization with an updated version of the manuscript. We are also working on additional swap lines (for example T2A-GAL4). Regarding submission of the materials to relevant databases, we are in the process of depositing the plasmids on Addgene. We plan to deposit the swap lines and other toolkit stocks (new hs-Flp, vas-int lines as well as pBac transposase lines) at the VDRC or BDSC. To make the tagged fly lines viable for distribution via the VDRC, we are working to increase their numbers, and we plan to publish them separately as a resource, where we also plan to characterize the expression of more transcription factors and their isoforms in greater detail.

    The Introduction should better reflect the current state of the field, including explicit comparison with MiMIC and CRIMIC.

    The introduction would benefit from a clearer distinction between transgene-based approaches that introduce additional gene copies and true CRISPR-mediated knock-ins at the endogenous locus. As it stands, the discussion of prior methods does not sufficiently acknowledge that CRISPR-based knock-in is already the standard approach in Drosophila, and that the individual techniques employed in ExTaSy are well established. Notably, the MiMIC and CRIMIC systems (Nagarkar-Jaiswal et al., 2015; Li-Kroeger et al., 2018), which also support RMCE-based tag exchange at endogenous loci and for which large collections of lines are already publicly available, are not adequately discussed. These are arguably the closest comparators to ExTaSy, and the authors should explicitly address how their approach differs from and offers advantages over this existing framework, particularly given that MiMIC/CRIMIC insertions can also tag internal sites and thus avoid some of the terminus-specific complications described here.

    __Response: __We will expand the introduction and the discussion to give more reference to other resources for endogenously and exogenously tagged genes in Drosophila and compare ExTaSy in greater detail with other methods, highlighting advantages and disadvantages of each and making clear that RMCE-based tag exchange and marker removal are not novel inventions.

    Minor Comment

    The labelling of sgRNA target sites in Figure 1 is inaccurate and should be corrected.

    In Figure 1, the sgRNA target sites are annotated with triangles labelled "PAM synth." The presence of a PAM is necessary but not sufficient to define a target site; the label should therefore be changed to "target site" or an equivalent term. Additionally, the Methods section incorrectly expands PAM as "primary adjacent motif"; the correct expansion is "protospacer adjacent motif."

    __Response: __The labelling in Figure 1 will be changed and the PAM abbreviation corrected.

    Could the fly crossing scheme in Figure S3 be simplified?

    In the scheme in Fig. S3 the second step seems to be intended to introduce the hs-Flp and vase-Int transgenes. Would it not be possible to already incorporate the Integrase into the swap fly line when it is made and the hs-Flp into the ExTaSy line, thereby saving one generation?

    __Response: __This would in principle be possible; however, we prefer to keep the lines “clean” in case a tag exchange is not desired, and so this would require an initial crossing step. We therefore prefer the crossing scheme as it is.

    Figure 1F has no call out in the main text.

    __Response: __This will be corrected.

    Line 155: What was the reason for the low survival rate? Is this likely to be indicative of a problem during marker removal, or a stochastic event as not all fly crosses are always productive (bad food, early death of flies, etc.)?

    __Response: __This was a stochastic event. The fly line we used for expression of piggyBac transposase (BDSC_8285) is generally not growing well, and we could only use one eighth of all offspring to ensure correct segregation. We will make this clear in the text.

    Line 160: What is the N number of "all cases"?

    __Response: __This will be changed to “We performed Sanger sequencing for one established line for each of the 17 loci and confirmed clean excision of the piggyBac sites in all cases.”

    Scale bars are missing in Fig. 3g,h.

    __Response: __These will be included.

    Line 219: The labeling of the panels got mixed up. Panel F does not show an immunostaining.

    __Response: __The labeling will be corrected.

    Line 226 and Fig. 3h: It is unclear what area is shown in the inlay. The overview image highlights three POIs, but none seem to fit the inlay.

    __Response: __The images were indeed misleading as the inlay did not show a magnification of the same focal plane. We will show the inlay together with the overview of the corresponding focal plane as part of Supplementary Figure 5 and will amend the text accordingly.

    Line 233: Why was the transgenic marker not removed? The authors want to highlight the easy and advantage of marker removal, so leaving in the marker is an odd choice.

    __Response: __In this case, we observed that flies become homozygous even with the marker, so we assumed that a marker removal would not be necessary. We are currently performing additional experiments to remove the marker and repeat the staining, which we will submit with a revised version of the manuscript.

    Line 250: Why was only one isoform of hth tagged? Without a rational this seems to be an odd choice, in particular since the authors seem to suggest in the introduction (Line 38) that a disadvantage of previous technologies is the tagging of only selected isoforms.

    __Response: __While expanding the introduction (see comment #4), we will also rephrase it to highlight that current CRISPR-based methods (MiMIC and CRIMIC) are designed to tag all isoforms simultaneously or select isoforms, whereas overexpression constructs are limited to one isoform. In contrast, ExTaSy allows tagging of all isoforms that share a terminus. We will emphasize advantages and disadvantages in the discussion. In the case of hth, three different C-termini are annotated, and we are currently performing experiments to also tag the other termini and co-stain them with Ubx. We will submit the results in a revised version of the manuscript.

    Reviewer #1 (Significance (Required)):

    SIGNIFICANCE

    ExTaSy assembles a set of well-established tools, namely CRISPR-mediated HDR, piggyBac-based marker excision, and φC31-mediated RMCE, into a unified, single-vector framework for endogenous protein tagging in Drosophila. The individual components have all been described and are in routine use in the field; the conceptual advance is therefore limited. Nevertheless, the integration of these features into a streamlined platform with accompanying automated design software represents a practical contribution that is likely to be of genuine utility to the Drosophila community, particularly for laboratories without specialist transgenesis infrastructure.

    The possibility of tag exchange by fly crossing is the most distinctive feature of the system. However, as discussed above, this is currently demonstrated at only five loci with a single replacement tag, which limits the conclusions that can be drawn about its generality. More broadly, ExTaSy employs well-proven strategies throughout, which is a source of reliability but also means that the study does not incorporate more recent developments in the field. For example, approaches based on single-strand annealing, such as the recently described Seed/Harvest system (Aguilar et al., 2024), can achieve entirely scarless marker removal and thus circumvent the TTAA scar left by piggyBac excision, a limitation the authors themselves acknowledge may reduce expression at modified N-terminal loci. Similarly, the current system is restricted to N- and C-terminal tagging. Given that the goal of endogenous tagging is to minimally perturb protein function, and given the now widespread availability of high-quality protein structure predictions for the Drosophila proteome, a modern tagging platform might be expected to use structural modelling to identify optimal insertion sites irrespective of their location. These are not oversights that diminish the practical value of the current work, but highlight that this study does not always operate at the cutting edge of method development in this area. A brief discussion of these more recent developments in the context of ExTaSy's design choices would usefully situate the work within the broader landscape and help readers understand both what the system offers today and where improvements are likely to come from.

    __Responses: __

    • As stated above, we are currently performing experiments to further validate the tag exchange.
    • Regarding the SEED/Harvest system, we have considered this; however, this would leave both flanking attP/attB sites at the genomic locus rather than only the site between the tag and the CDS. Both sites would have to be incorporated into the CDS or they would leave an even bigger scar. Additionally, since SEED/Harvest relies on micro-homology between two tag halves, it would require removal of the transgenesis marker before tagged lines become usable. Our system is advantageous in that C-terminally tagged lines can usually be used immediately. However, we will refer to the paper by Aguilar et al. and discuss how a similar system could be incorporated into ExTaSy.
    • Regarding structure-function predictions, these could be incorporated into the bioinformatic pipeline. It would then be possible to modify ExTaSy to introduce tags internally together with a SEED/Harvest-like modification. We will include this in the discussion.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary

    Hubbert et al. describes ExTaSy (Exchangeable Tagging System), a method for endogenous protein tagging in fruitflies. The technique attempts to address some limitations of current tagging strategies, such as non-physiological expression from transgenes, disruption of the target gene, and limited usefulness of a single tag type. The basic approach is not novel, rather it effectively incorporates ideas from several previously published methods:

    • Crispr-based release of the HDR donor from the backbone in vivo (Kanca et al., 2019 and 2021).
    • PBac scarless tagging (flycrisprdesign)
    • In vivo RMCE to swap out tags (Nagarkar-Jaiswal et al., 2015) Although not novel, the authors show the completeness and effectiveness of the approach. They were able to tag genes across multiple chromosomes, with knock-in rates comparable to other approaches, and demonstrate tag swapping through RMCE. Overall, this work introduces a versatile and modular platform that combines several previous innovations into a single effective package.

    Major comments

    1.The manuscript would benefit from a more upfront discussion of how ExTaSy relates to existing methods. As currently written, the implies a higher degree of novelty than is warranted, since ExTaSy combine several previously established approaches, including, as already noted. While this is valuable, the authors should more clearly acknowledge in the abstract and introduction that the primary advance is the unification and streamlining of these existing technologies into a single platform, rather than the introduction of fundamentally new components.

    __Response: __While we did cite most of the publications mentioned by the reviewer, we will make clearer that our system combines several previously established Drosophila systems and is not per se a novel invention. We will expand the introduction and discussion to reflect this and cite additional publications.

    2.Comparison to prior systems. The manuscript should include a direct comparison to existing tagging pipelines. For example: What practical steps are eliminated relative to prior approaches? Does ExTaSy reduce the number of injections or constructs required? How does the workflow differ in terms of time, cost, or technical expertise? This is vaguely addressed in the discussion, but more specific and clear comparisons would improve things for the reader who is trying to decide which method to use. For example, how does this strategy directly compare with the protein trap alleles described in Kanca et al., 2022? This could be done as a supplemental table.

    __Response: __A similar concern has been raised by reviewer #1 (comment #4). We will expand the introduction and the discussion to compare ExTaSy in more detail with other methods, highlighting advantages and disadvantages of each.

    3.Only 4 successful RMCE swaps are presented. This is too few to make a confident conclusion about the efficiency. The authors should do at least 4 more and include negative data.

    __Response: __A similar point has been made by reviewer #1 (comment #3). We are in the process of expanding the number of fly stocks for which tags have been exchanged and will be able to provide a more rigorous characterization with an updated version of the manuscript.

    4.Some discussion of the potential limitations of the linker from the residual att sites is needed.

    __Response: __We will include this in the discussion.

    Minor comments

    1.It would be helpful to include a workflow overview figure summarizing the full pipeline.

    __Response: __We will include such a figure in the supplement.

    2.Line 124: Most genes we tagged at the C-terminus were homozygous viable, indicating limited detrimental effects. Need to include the numbers? What is "most genes."

    __Response: __We will include these numbers in the text.

    3.Briefly explain how the tested genes were selected (e.g., random, representative, biased toward certain classes), as this could affect interpretation of generalizability. If most of the genes are essential for viability, this makes the viability of tagged lines more impressive.

    __Response: __This is an excellent suggestion, and we thank the reviewer for pointing this out. We have mainly tagged genes that are relevant for work in our labs and for collaborators, focusing almost entirely on transcription factor-encoding genes that are largely essential for normal development. We will include a brief discussion of this.

    Reviewer #2 (Significance (Required)):

    Significance

    1.General assessment: This study presents ExTaSy, a practical and well-executed platform for endogenous protein tagging in Drosophila. Its main strength is the integration of multiple existing technologies into a streamlined workflow that enables tagging, marker removal, and tag swapping. The system is clearly functional and broadly applicable. However, the conceptual novelty is limited, and the manuscript should more explicitly frame the work as an engineering advance. Tagging and RMCE efficiencies are moderate.

    2.Advance: ExTaSy represents a technical advance that combines CRISPR HDR tagging, piggyBac scarless editing, and RMCE into a single platform. The biggest improvement is the ability to tag once and flexibly swap tags via crosses, reducing the need for repeated genome engineering. This extends existing methods by improving experimental flexibility.

    3.Audience: This work will primarily interest a specialized audience in Drosophila genetics, CRISPR technologies, and functional genomics, with broader relevance to researchers developing tagging systems in other model organisms.

    4.Field of expertise: CRISPR screening, Drosophila genetics, functional genomics. No limitations on my ability to evaluate.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This methods paper is targeting the long-standing ambition of how to most efficiently tag proteins at the endogenous gene locus in Drosophila. Since the invention of CRISPR-Cas9 many genes have been successfully modified in Drosophila, but the community is still lacking a large collection of tagged proteins under endogenous control made with the same method.

    This manuscript is using a small tag, 3xHA, which supposedly is easier to integrate, and the design allows to then swap the tag with larger fluorescent tags, solely by fly crossing. Then, the dsRed or white markers, allowing identification, can be removed with a biggybac recombinase leaving only a small scar. However, attP/B/R scars do remain. Design and cloning appear straightforward. Overall, this is an interesting strategy.

    However, the manuscript falls short in really describing the resource, apart from the cloning design. A more rigorous analysis of a number of lines should be presented to better judge if the strategy practically works. It is quite disappointing to see that only 2 or 3 genes/proteins were analysed here in a bit more detail. This does not sound like a very straightforward resource that aims to go large scale.

    Major comments:

    The important novelty here is not only the design that allows high-throughput cloning but more importantly that the tagged lines are actually correct and functional. To present this better, I suggest to rearrange Figure 1 to show the flow: 65 constructs cloned, 41 "successfully" inserted. Of how many the dsRed marker was removed, of how many expression or function was tested? Hence the reader knows about the current state of the resource. These numbers would be informative to have in the abstract, too.

    __Response: __We will include these numbers in the abstract. Reviewer 2 asked for an overview figure of the workflow, which we will include as a supplementary figure, where we can also include numbers as suggested by this reviewer.

    The 41 tagged gene insertions need at least some basic characterisation to verify that they are at the correct place or make a functional protein. Which genes were chosen? I do not see 41 genes tagged in the table provided. I supposed the N-terminal tags should initially be loss of function. Are the N-term lines lethal when inserted in an essential gene? Again, this could be shown in an overview, instead by a non-quantitative statement in the text.

    __Response: __We have verified the insertion site of the lines with genotyping PCR. We will include a table to show in more detail which genes were tagged at which terminus, and which protein isoforms are captured by the respective tag.

    How many of the 41 tagged proteins are functional? The authors only provide information on Ubx-3xHA (functional) and Mef2-3xHA (non-functional), which I find weak.

    __Response: __We will include this information in the table mentioned in the above comment.

    Stainings are only shown for 2 proteins, Ubx-GFP and Exd-3xHA. How about the others?

    __Response: __We are currently in the process of using ExTaSy to establish a library of tagged fly lines, which we intend to characterize in more detail and publish separately. For the current manuscript, we prefer to focus on the methodology of the tagging system itself.

    I am not sure about how to calculate the transgenesis rates, but strictly speaking to ones that did not result in an insertion should also be counted for the statistics, I guess.

    __Response: __There is indeed no commonly agreed upon way to calculate these rates, and it is done differently in different publications. We felt that metrics that discriminate between the overall success rate (i.e., all those injections that lead to transgenics) and the success rate within successful injections would be most useful. We will try to make clear in the text where we refer to all attempts and where we exclusively refer to the successful ones.

    Minor comments:

    The introduction states that ExTaSy would tag all isoforms of genes. However, I find this an overstatement, as for complex genes tagging at the one place cannot always label all isoforms, see the Hth line generated here (Iso E).

    __Response: __This was indeed badly phrased and we will correct the wording also in response to reviewer #1 comment #14 to reflect that overexpression constructs are limited to a specific isoform, whereas ExTaSy enables simultaneous tagging of all isoforms that share a terminus.

    Why does it matter on which chromosome the target gene is? This can be moved to supplement. I would rather like to know what the genes are.

    __Response: __We presume that the reviewer refers to Figure 1, where we show the success rates for individual chromosomes. We felt that the lower success rate for injections targeting gene on chr3 (which is, as we describe, due to lower survival of the injection line) warranted this separation by chromosome. As stated above, we will include a list of tagged genes as a table.

    **Referees cross-commenting**

    I agree with the 2 other reviewer's points. In particular that the knock-in lines need better verifications. This was also my major point.

    __Response: __As also stated for reviewer #1 comment #1, we have now begun to run whole-fragment PCRs for all loci to investigate this further and will report the results in a revised version of the manuscript.

    Reviewer #3 (Significance (Required)):

    The methodology presented here is per se not really new. The 3xP3-dsRed eye marker is standard, its removal by biggbac transposase has been done before and RMCE to change the tagging cassettes with attP/B is done since many years. The latter has the disadvantage to not be seamless, as one attR site remains, which is translated, the other attR site remains in the 5'- or 3'-UTR, which can have an effect. U6-driven sgRNA expression is also standard.

    __Response: __We will make clearer that our system combines several previously established Drosophila systems and is not per se a novel invention. We will expand the introduction and discussion to reflect this and cite additional publications.

    The design includes the sgRNA and the HDR template cassette in a single vector, which is smart and makes cloning straight forward. Again, the paper would be stronger if the list of all cloned clones would be listed (are 65 all that were clones or all that were injected?

    __Response: __We will include this as a table.

    As the authors do not rigorously test the function of the tagged genes, it is hard to judge how valuable the pipeline is. This can be easily solved by providing more data that support the easy, high-throughput exchange tagging pipeline that produces tagged Drosophila lines that are useful to the community.

    __Response: __As stated above, we plan to publish a more detailed analysis of tagged lines as a separate resource paper. We will state in the manuscript which lines were homozygous viable before and after marker removal, which gives at least an indication of whether the tagged protein is functional.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This methods paper is targeting the long-standing ambition of how to most efficiently tag proteins at the endogenous gene locus in Drosophila. Since the invention of CRISPR-Cas9 many genes have been successfully modified in Drosophila, but the community is still lacking a large collection of tagged proteins under endogenous control made with the same method. This manuscript is using a small tag, 3xHA, which supposedly is easier to integrate, and the design allows to then swap the tag with larger fluorescent tags, solely by fly crossing. Then, the dsRed or white markers, allowing identification, can be removed with a biggybac recombinase leaving only a small scar. However, attP/B/R scars do remain. Design and cloning appear straightforward. Overall, this is an interesting strategy. However, the manuscript falls short in really describing the resource, apart from the cloning design. A more rigorous analysis of a number of lines should be presented to better judge if the strategy practically works. It is quite disappointing to see that only 2 or 3 genes/proteins were analysed here in a bit more detail. This does not sound like a very straightforward resource that aims to go large scale.

    Major comments:

    1. The important novelty here is not only the design that allows high-throughput cloning but more importantly that the tagged lines are actually correct and functional. To present this better, I suggest to rearrange Figure 1 to show the flow: 65 constructs cloned, 41 "successfully" inserted. Of how many the dsRed marker was removed, of how many expression or function was tested? Hence the reader knows about the current state of the resource. These numbers would be informative to have in the abstract, too.
    2. The 41 tagged gene insertions need at least some basic characterisation to verify that they are at the correct place or make a functional protein. Which genes were chosen? I do not see 41 genes tagged in the table provided. I supposed the N-terminal tags should initially be loss of function. Are the N-term lines lethal when inserted in an essential gene? Again, this could be shown in an overview, instead by a non-quantitative statement in the text.
    3. How many of the 41 tagged proteins are functional? The authors only provide information on Ubx-3xHA (functional) and Mef2-3xHA (non-functional), which I find weak.
    4. Stainings are only shown for 2 proteins, Ubx-GFP and Exd-3xHA. How about the others?
    5. I am not sure about how to calculate the transgenesis rates, but strictly speaking to ones that did not result in an insertion should also be counted for the statistics, I guess.

    Minor comments:

    1. The introduction states that ExTaSy would tag all isoforms of genes. However, I find this an overstatement, as for complex genes tagging at the one place cannot always label all isoforms, see the Hth line generated here (Iso E).
    2. Why does it matter on which chromosome the target gene is? This can be moved to supplement. I would rather like to know what the genes are.

    Referees cross-commenting

    I agree with the 2 other reviewer's points. In particular that the knock-in lines need better verifications. This was also my major point.

    Significance

    The methodology presented here is per se not really new. The 3xP3-dsRed eye marker is standard, its removal by biggbac transposase has been done before and RMCE to change the tagging cassettes with attP/B is done since many years. The latter has the disadvantage to not be seamless, as one attR site remains, which is translated, the other attR site remains in the 5'- or 3'-UTR, which can have an effect. U6-driven sgRNA expression is also standard. The design includes the sgRNA and the HDR template cassette in a single vector, which is smart and makes cloning straight forward. Again, the paper would be stronger if the list of all cloned clones would be listed (are 65 all that were clones or all that were injected?

    As the authors do not rigorously test the function of the tagged genes, it is hard to judge how valuable the pipeline is. This can be easily solved by providing more data that support the easy, high-throughput exchange tagging pipeline that produces tagged Drosophila lines that are useful to the community.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Hubbert et al. describes ExTaSy (Exchangeable Tagging System), a method for endogenous protein tagging in fruitflies. The technique attempts to address some limitations of current tagging strategies, such as non-physiological expression from transgenes, disruption of the target gene, and limited usefulness of a single tag type. The basic approach is not novel, rather it effectively incorporates ideas from several previously published methods:

    • Crispr-based release of the HDR donor from the backbone in vivo (Kanca et al., 2019 and 2021).
    • PBac scarless tagging (flycrisprdesign)
    • In vivo RMCE to swap out tags (Nagarkar-Jaiswal et al., 2015) Although not novel, the authors show the completeness and effectiveness of the approach. They were able to tag genes across multiple chromosomes, with knock-in rates comparable to other approaches, and demonstrate tag swapping through RMCE. Overall, this work introduces a versatile and modular platform that combines several previous innovations into a single effective package.

    Major comments

    1.The manuscript would benefit from a more upfront discussion of how ExTaSy relates to existing methods. As currently written, the implies a higher degree of novelty than is warranted, since ExTaSy combine several previously established approaches, including, as already noted. While this is valuable, the authors should more clearly acknowledge in the abstract and introduction that the primary advance is the unification and streamlining of these existing technologies into a single platform, rather than the introduction of fundamentally new components. 2.Comparison to prior systems. The manuscript should include a direct comparison to existing tagging pipelines. For example: What practical steps are eliminated relative to prior approaches? Does ExTaSy reduce the number of injections or constructs required? How does the workflow differ in terms of time, cost, or technical expertise? This is vaguely addressed in the discussion, but more specific and clear comparisons would improve things for the reader who is trying to decide which method to use. For example, how does this strategy directly compare with the protein trap alleles described in Kanca et al., 2022? This could be done as a supplemental table. 3.Only 4 successful RMCE swaps are presented. This is too few to make a confident conclusion about the efficiency. The authors should do at least 4 more and include negative data. 4.Some discussion of the potential limitations of the linker from the residual att sites is needed.

    Minor comments

    1.It would be helpful to include a workflow overview figure summarizing the full pipeline. 2.Line 124: Most genes we tagged at the C-terminus were homozygous viable, indicating limited detrimental effects. Need to include the numbers? What is "most genes." 3.Briefly explain how the tested genes were selected (e.g., random, representative, biased toward certain classes), as this could affect interpretation of generalizability. If most of the genes are essential for viability, this makes the viability of tagged lines more impressive.

    Significance

    1.General assessment: This study presents ExTaSy, a practical and well-executed platform for endogenous protein tagging in Drosophila. Its main strength is the integration of multiple existing technologies into a streamlined workflow that enables tagging, marker removal, and tag swapping. The system is clearly functional and broadly applicable. However, the conceptual novelty is limited, and the manuscript should more explicitly frame the work as an engineering advance. Tagging and RMCE efficiencies are moderate. 2.Advance: ExTaSy represents a technical advance that combines CRISPR HDR tagging, piggyBac scarless editing, and RMCE into a single platform. The biggest improvement is the ability to tag once and flexibly swap tags via crosses, reducing the need for repeated genome engineering. This extends existing methods by improving experimental flexibility. 3.Audience: This work will primarily interest a specialized audience in Drosophila genetics, CRISPR technologies, and functional genomics, with broader relevance to researchers developing tagging systems in other model organisms. 4.Field of expertise: CRISPR screening, Drosophila genetics, functional genomics. No limitations on my ability to evaluate.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Hubbert and colleagues describe ExTaSy, a CRISPR-Cas9-based platform for the endogenous tagging of proteins in Drosophila melanogaster. The system combines several established molecular tools into a single-vector framework: homology-directed repair (HDR) for the insertion of a 3XHA tag at the endogenous locus, piggyBac transposase-mediated near-scarless removal of a transgenic selection marker, and φC31 integrase-mediated recombination-mediated cassette exchange (RMCE) for subsequent tag swapping. The authors demonstrate the system across a set of 65 genomic loci and provide a bioinformatic pipeline to automate guide RNA and homology arm design.

    Major Comments

    1. Validation of knock-in lines is inadequate and does not reflect current standards in the field.

    The authors state that correct insertions were confirmed using "two PCRs per inserted fragment done with primers binding to the 5' and 3' ends of the inserted DNA and corresponding gene-specific validation primers." This strategy is well known to produce false positives, as it cannot distinguish correctly targeted single-copy integrants from concatemeric insertions at the target locus (e.g. Skryabin et al., 2020). The current standard for validating CRISPR-mediated knock-ins requires PCR amplification using primers that anneal outside the homology arms and span the entire inserted cassette. These reactions must be performed under conditions that minimise the formation of PCR chimeras, specifically low cycle numbers and use of a high-processivity polymerase. The authors should either provide data from such experiments for their characterised lines, or clearly acknowledge this limitation and qualify their efficiency estimates accordingly (see related point 2 below).

    1. Reported efficiency metrics do not adequately distinguish correctly targeted integrants from marker-positive flies.

    A related concern is that many of the efficiency parameters reported in the manuscript appear to be based solely on the detection of the marker cassette. The 63.1% overall success rate, for example, seemingly reflects the recovery of DsRed-positive flies rather than of sequence validated, single-copy, on-target integrants. These are fundamentally different quantities, with only the latter being of practical value for the users of the described technique. The authors should either provide data that properly accounts for correct integration, or more carefully define what each reported metric represents and explicitly acknowledge the limitations of using marker presence as a proxy for successful knock-in.

    1. The characterisation of tag exchange requires expansion or more careful framing of its scope.

    The possibility of exchanging tags through fly crosses rather than repeated microinjections is, in the view of this reviewer, the most practically useful feature of ExTaSy and the aspect most likely to drive community adoption. It is therefore important that this feature is characterised with sufficient rigour to allow prospective users to assess its reliability. In the current manuscript, tag exchange has been demonstrated at only five loci using a single replacement tag (sfGFP). The dataset includes one outright failure (the Met C-terminus) and one instance of an unexpected 9 bp insertion at the recombination site, leaving the success rates and failure modes across a broader range of loci and tags uncharacterised. The authors should either expand the tag exchange experiments to cover a more representative set of conditions, or frame the current data explicitly as a proof of concept and limit their conclusions about the practical utility of tag exchange accordingly. In either case, the value of this work to the community would be substantially increased if a collection of donor lines carrying the most commonly used tags for different applications, as the authors themselves enumerate in the Discussion, were generated and deposited at a public stock centre such as the VDRC concurrent with publication. On this note, it is also worth flagging that at present the plasmids described in this study have not yet been deposited at Addgene or the European Plasmid Repository, and that fly lines are available only on request. For a methods paper aimed at community adoption, deposition of reagents in publicly accessible repositories at the time of publication is the expected standard.

    1. The Introduction should better reflect the current state of the field, including explicit comparison with MiMIC and CRIMIC.

    The introduction would benefit from a clearer distinction between transgene-based approaches that introduce additional gene copies and true CRISPR-mediated knock-ins at the endogenous locus. As it stands, the discussion of prior methods does not sufficiently acknowledge that CRISPR-based knock-in is already the standard approach in Drosophila, and that the individual techniques employed in ExTaSy are well established. Notably, the MiMIC and CRIMIC systems (Nagarkar-Jaiswal et al., 2015; Li-Kroeger et al., 2018), which also support RMCE-based tag exchange at endogenous loci and for which large collections of lines are already publicly available, are not adequately discussed. These are arguably the closest comparators to ExTaSy, and the authors should explicitly address how their approach differs from and offers advantages over this existing framework, particularly given that MiMIC/CRIMIC insertions can also tag internal sites and thus avoid some of the terminus-specific complications described here.

    Minor Comment

    1. The labelling of sgRNA target sites in Figure 1 is inaccurate and should be corrected.

    In Figure 1, the sgRNA target sites are annotated with triangles labelled "PAM synth." The presence of a PAM is necessary but not sufficient to define a target site; the label should therefore be changed to "target site" or an equivalent term. Additionally, the Methods section incorrectly expands PAM as "primary adjacent motif"; the correct expansion is "protospacer adjacent motif."

    1. Could the fly crossing scheme in Figure S3 be simplified?

    In the scheme in Fig. S3 the second step seems to be intended to introduce the hs-Flp and vase-Int transgenes. Would it not be possible to already incorporate the Integrase into the swap fly line when it is made and the hs-Flp into the ExTaSy line, thereby saving one generation?

    1. Figure 1F has no call out in the main text.
    2. Line 155: What was the reason for the low survival rate? Is this likely to be indicative of a problem during marker removal, or a stochastic event as not all fly crosses are always productive (bad food, early death of flies, etc.)?
    3. Line 160: What is the N number of "all cases"?
    4. Scale bars are missing in Fig. 3g,h.
    5. Line 219: The labeling of the panels got mixed up. Panel F does not show an immunostaining.
    6. Line 226 and Fig. 3h: It is unclear what area is shown in the inlay. The overview image highlights three POIs, but none seem to fit the inlay.
    7. Line 233: Why was the transgenic marker not removed? The authors want to highlight the easy and advantage of marker removal, so leaving in the marker is an odd choice.
    8. Line 250: Why was only one isoform of hth tagged? Without a rational this seems to be an odd choice, in particular since the authors seem to suggest in the introduction (Line 38) that a disadvantage of previous technologies is the tagging of only selected isoforms.

    Significance

    ExTaSy assembles a set of well-established tools, namely CRISPR-mediated HDR, piggyBac-based marker excision, and φC31-mediated RMCE, into a unified, single-vector framework for endogenous protein tagging in Drosophila. The individual components have all been described and are in routine use in the field; the conceptual advance is therefore limited. Nevertheless, the integration of these features into a streamlined platform with accompanying automated design software represents a practical contribution that is likely to be of genuine utility to the Drosophila community, particularly for laboratories without specialist transgenesis infrastructure.

    The possibility of tag exchange by fly crossing is the most distinctive feature of the system. However, as discussed above, this is currently demonstrated at only five loci with a single replacement tag, which limits the conclusions that can be drawn about its generality. More broadly, ExTaSy employs well-proven strategies throughout, which is a source of reliability but also means that the study does not incorporate more recent developments in the field. For example, approaches based on single-strand annealing, such as the recently described Seed/Harvest system (Aguilar et al., 2024), can achieve entirely scarless marker removal and thus circumvent the TTAA scar left by piggyBac excision, a limitation the authors themselves acknowledge may reduce expression at modified N-terminal loci. Similarly, the current system is restricted to N- and C-terminal tagging. Given that the goal of endogenous tagging is to minimally perturb protein function, and given the now widespread availability of high-quality protein structure predictions for the Drosophila proteome, a modern tagging platform might be expected to use structural modelling to identify optimal insertion sites irrespective of their location. These are not oversights that diminish the practical value of the current work, but highlight that this study does not always operate at the cutting edge of method development in this area. A brief discussion of these more recent developments in the context of ExTaSy's design choices would usefully situate the work within the broader landscape and help readers understand both what the system offers today and where improvements are likely to come from.

  5. Version published to 10.64898/2026.01.16.699669 on bioRxiv