These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no ‘two-in-one’ KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at  Howevero rerio) . Alternarategies . Howeverrategies . FurtherloxP sequences have to be inserted at two separate locations  or the NHEJ pathway for repairing DNA double-strand breaks , and NHEocations . Alternaocations . Neverthtbx5a and kctd10 loci, resulting in a germline transmission efficiency as high as 56% after the preselection of F0 embryos showing correct expression of a fluorescent reporter. Under normal conditions, these KI alleles enable simultaneous expression of a fluorescent reporter together with the target gene, displaying a gene-labeling effect. After the injection of Cre mRNA, the expression and function of the target gene as well as the reporter gene were disrupted, and conditional knockout was successfully achieved. We further improved our strategy by introducing a second fluorescent reporter into the dual-cassette donor, which could be used to conditionally generate and distinguish different alleles  by using different fluorescent reporters and, further, to distinguish different cells with different genotypes , allowing real-time mosaic analysis within the same tissue. This allele-tagging or geno-tagging strategy was demonstrated at the tbx5a and sox10 loci.Here, we report the development of a highly efficient method for achieving one-step heritable generation of dual-function alleles in zebrafish through targeted insertion induced by the Cas9/gRNA system, based on the special design of a dual-cassette-donor. The feasibility of this strategy was first demonstrated at the via the NHEJ pathway activated by the CRISPR/Cas system. For this purpose, the initial donor was designed to contain two functional cassettes, a positive one and a negative one and is therefore called a PoNe (positive-negative) donor  was designed to ensure complete disruption of target gene expression and function after removal of the Po-cassette by Cre-induced recombination. This is achieved by two levels of interruption of gene expression, leading to the early termination of either transcription or translation. For this purpose, the initial Ne-cassette consisted of two transcription termination signals (TTSs), the SV40 polyA (PA) sequence followed by BGH PA, which were followed by the sequence of the same splice acceptor site as in the Po-cassette and the exon immediately after the target intron, but engineered to contain a nonsense mutation (mutExon).To generate a dual-function allele through a single step of NHEJ-mediated KI, we developed a novel strategy based on the special design of a dual-cassette donor, in which CKO and gene labeling can be established simultaneously through one-step targeted insertion of the donor vector into an intron of the target gene e) donor . The posloxP-mediated removal of the Po-cassette, the two polyA signals (2PA) in the Ne-cassette could prematurely stop the transcription of the target gene, resulting in a short transcript encoding a truncated and nonfunctional protein, thus achieving CKO. If there is leaky transcription after the polyA signals, the mutExon sequence in the Ne-cassette will be transcribed and spliced into the mature mRNA, again resulting in the disruption of the function of the target gene, due to the existence of the premature stop codon in this exon.After the insertion of the PoNe donor into the intron of the target gene, the targeted locus will acquire the properties of both a conditional allele and a gene-tagging allele. Under normal conditions, that is in the absence of Cre recombinase, this locus serves a gene-tagging function, and the expression and function of the target gene remain normal; a wild-type protein and a fluorescent reporter protein will be produced concurrently, with the fluorescent reporter recapitulating the expression pattern of the endogenous target gene. After Cre/tbx5a locus  , dramatically affected KI efficiency in our experimental settings. In our initial attempts, we employed ethanol precipitation to purify both gRNAs used for injection and found no tdTomato expression in any injected embryos. In contrast, when injected with LiCl-purified gRNAs, more than 10% of the embryos showed the expected red fluorescent signal (hEMX1 site) purified by LiCl precipitation and the endogenous gene (tbx5a)-targeting gRNA purified by ethanol precipitation. However, no fluorescent signal could be detected with the other combination, that is the donor gRNA purified by ethanol precipitation and the tbx5a-targeting gRNA purified by LiCl precipitation  . After tobserved . Junctioget site . Surprist signal . Furtherpitation . These otbx5a PoR-Ne donor together with the Cas9/gRNA system into approximately 100 one-cell stage embryos, red fluorescent signals could be detected in 18 of the embryos. These founder (F0) embryos were separated into tdTomato-positive and tdTomato-negative groups and raised to adulthood for germline transmission analysis. Correct expression patterns of red fluorescent signals were detected in the F1 embryos derived from the outcrossing of 5 out of 9 F0 individuals grown from the tdTomato-positive embryos, with mosaicism ranging from 2.4% to 43.8%  transgenic background  and the highest expression in the atrioventricular canal (AVC)  . This phal (AVC) , indicattbx5a PoR-Ne donor KI alleles that we generated are not only gene-tagging alleles but also floxed conditional alleles. To evaluate whether conditional knockout could be achieved with these alleles, we isolated a tbx5a indel mutation with a 5 bp deletion in the third exon (Δ5tbx5a) (Δ5/Δ5tbx5a homozygote embryos showed severe heart and fin defects at 48 hpf (hours post-fertilization) , and their progeny were injected with Cre mRNA at the one-cell stage. As expected, some of the injected F1 embryos  showed distinct developmental defects of the heart and fins , while the normal ones initially presented the +/Δ5tbx5a, +/PoR-Netbx5a, or +/+tbx5a genotype   experiments were performed on 72 hpf embryos using primers T5qF and T5qR  . tbx5aΔ5ization) , which imutation . A tbx5aand fins . No red  allele) . To evaland T5qR  in exonsn’ state . No red expected . To mease allele . CollecttdTomato with tdGFP as the reporter at another zebrafish locus, kctd10  displayed germline transmission of the kctd10 PoG-Ne donor KI events  derived from two F0 individuals harboring two slightly different PoG-Ne insertions   , 25.9%  in its Ne-cassette. Using such donors, we were able to isolate stable integrations at the kctd10 locus and observed the correct expression pattern of tdGFP. However, after Cre mRNA injection, all the injected embryos resulting from the incrosses of such heterozygous F1 fish developed normally, although the green fluorescent signal was no longer detectable in most of the embryos, indicating the successful and efficient removal of the Po-cassette. These results suggest that the single SV40 PA signal might not be strong enough to efficiently stop target gene transcription; thus, either multiple copies of the transcription termination sequences are necessary, or the modified exon (mutExon) following the PA sequence might be indispensable for efficiently disrupting target gene expression in our strategy (data not shown).In the initial experiments, we used a donor with a single SV40 via one-step targeted insertion. To further improve our strategy and achieve more complex applications of genome editing, we developed a novel technique by designing a conditional dual-fluorescence reporter donor  can be labeled with different fluorescent proteins. This goal was achieved by adding a different fluorescent reporter in the original Ne-cassette following the sequence containing the intron (including the splice acceptor site) plus a partial exon immediately downstream of the intron target site, and linked by a 2A peptide, as in the Po-cassette. After the removal of the Po-cassette containing the first fluorescent reporter by the Cre/loxP system, the second fluorescent reporter in the Ne-cassette will be transcribed together with the preceding intron and exon sequences and spliced into the incomplete mRNA of the target gene, thus achieving both target gene mutation (due to the incomplete coding sequence) and labeling of the mutated allele, simultaneously leading to the destruction of the target gene’s function and the allele-tagging switch. Under this strategy, the functionally normal and defective alleles can be easily distinguished by using two different fluorescent reporters. Note that the wild-type exon sequence was used in the Ne-cassette in this case since we would like to maintain normal transcription and translation of the fusion mRNA, which contains the incomplete target gene cDNA followed by a 2A-linked complete sequence encoding the fluorescent reporter  together with the zCas9 mRNA and corresponding gRNAs, red fluorescent signals could be detected in 16 of 124 normally developed F0 embryos. The ratio of embryos showing red fluorescent signals was comparable with that in the previous tbx5a PoR-Ne donor KI experiment , indicating the successful integration of the donor in somatic cells , and germline transmission of the tbx5a PoR-NeG donor geno-tagging alleles was detected and confirmed in two F0 individuals (#2 and #42)  . In contPoR-NeGtbx5a. We injected Cre mRNA into the F1 progeny from F0 #42 outcrossed with wild-type zebrafish and observed successful switching of the fluorescent signals , showing heart-specific expression of both ECFP and 4-hydroxyltamoxifen (4-HT)-inducible zebrafish codon-optimized T2CreER. After crossing this transgenic fish with PoR-NeG/PoR-NeGtbx5a adults, the embryos were collected and separated into two groups. Then 4-HT was added to one group from 5 hpf to 48 hpf. Green fluorescent signals were detected in the hearts of 56 hpf ECFP-positive embryos, indicating the successful switching of tdTomato to tdGFP expression in some cells  . We first coinjected the sox10 geno-tagging donor with the zCas9 system into one-cell-stage zebrafish embryos, after which approximately half of the embryos were injected again with 25 pg of Cre mRNA in a single cell at the 4 cell stage. As expected, we detected the expression of two different fluorescent reporters in otic vesicles as well as in cranial and trunk neural crest cells in some embryos (29/126) injected with Cre mRNA   to green (tdGFP) was observed in all the injected fluorescent embryos  of the injected embryos exhibited extensive loss of pigmentation at 48 hpf, recapitulating the phenotype reported previously for the sox10 mutant colourless  . In addiamGolden  for the Cre mRNA . In someCre mRNA , indicat103/152) . NHEJ-me103/152) . The 103to 50.0% . Positiv embryos , indicatlourless , and allG allele . Genotyp0 allele . qRT-PCRcassette . The traG allele . Taken ttdTomato or tdGFP) in the positive-cassette of the donor ; (iii) differential labeling of two different alleles  to achieve a geno-tagging effect via the differential labeling of the two cassettes (Po- vs Ne-cassette) in the donor with two different fluorescent reporters. This advanced strategy enables the conditional switching of the fluorescent reporters and, thus, the discrimination of cells with three different genotypes . We successfully generated such conditional coupled geno-tagging alleles at the tbx5a and sox10 loci in zebrafish and demonstrated the fluorescent reporter switch in response to Cre recombinase treatment. As the activity of Cre recombinase can easily be manipulated , genetic mosaicism of target tissues could be generated through conditional knockout. However, previously reported KI techniques usually cannot distinguish cells or tissues with different genotypes, which limits the in-depth study of gene functions. With simple modifications of our dual-cassette donor system, we have established a novel geno-tagging technique that allows conditional knockout and geno-tagging switching to occur simultaneously in the same allele. Fluorescence label switching, together with conditional gene knockout, not only allows the real-time labeling and discrimination of knockout cells or tissues from normal ones to trace and compare their fates in vivo but can even go further, potentially labeling different alleles with different fluorescent reporters. In this manner, one can easily determine and follow the genotype of each allele and each cell.In summary, we reported a highly efficient knockin strategy based on the special design of multipurpose dual-cassette PoNe donors and NHEJ-mediated targeted insertion, facilitated by the CRISPR/Cas system. Three levels of complex genome modifications could be achieved in zebrafish by using this method: (i) one-step generation of conditional knockout alleles through targeted insertion; (ii) simultaneous fluorescent labeling of CKO alleles to achieve a gene tagging effect as well as labeling of the cells expressing the target gene, through the incorporation of an in-frame fluorescent reporter gene  for stronger transcriptional termination for the sox10 locus. However, there are potential disadvantages of employing two 8xPA sequences, each containing eight tandem repeats of the ~160 bp polyA signal sequence, concerning stability. The repeats within each 8xPA sequence might be reduced during expansion and/or passaging to the next generations of the PoR-NeGsox10 allele; on the other hand, the sequence flanked by the two 8xPA sequences (containing the downstream loxP and Ne-cassette) might be lost due to occasional recombination of the two 8xPA sequences, leading to the abolition of the CKO and fluorescence-switch effects. Practically, since different target genes may have very different expression patterns and regulation properties, for each particular target gene, we recommend beginning with the construction of the donor with 2PA, then testing its effect in founder embryos to determine whether this is sufficient to stop the expression of the second fluorescent reporter, and introducing more polyA signals when necessary.In addition, there is another potential advantage of the geno-tagging strategy due to the special design of the Ne-cassette in the donor. While the original single-fluorescent-reporter version of the dual-cassette donor might trigger genetic compensation since it contains a cription . Althougtbx5a PoR-Ne KI fish line, approximately 15% of F0 embryos showed the expected tdTomato expression after the injection of the donor together with the Cas9/gRNA system. More than 50% (5/9) of these fluorescent reporter-positive F0 fish successfully transmitted the KI alleles to their progeny. In contrast, no germline transmission events were identified in the 40 founders negative for the somatic cell expression of tdTomato. Similarly, approximately half (10/21) of the embryonic stage preselected founders showed germline transmission of the sox10 geno-tagging KI alleles. For the screening of tbx5a PoR-NeG geno-tagging KI fish, we preselected adult fish for somatic cell integration by junction PCR of fin clips and found that 6 out of 48 founders were positive for donor insertions in this somatic tissue. Among these founders, 2 out of 5 positive founders were also positive for germline transmission. Not surprisingly, we also screened for germline transmission in 20 founders negative by fin clip junction PCR and found that none of them transmitted a KI donor to their offspring. In summary, to achieve efficient recovery of germline-transmissible KI alleles, we strongly recommend the preselection of F0 embryos according to fluorescent signals or adults on the basis of fin clip junction PCR and then screening only these founders in germline transmission analysis.Germline transmission is crucial for the successful establishment of stable KI transgenic fish lines. Interestingly, our results indicate a strong correlation between the efficient germline transmission and correct insertion of KI donors in somatic tissues. We observed high-efficiency germline recovery of the KI events after preselection for mosaic founders that acquired the correct donor insertions in somatic tissues by selecting either embryos on the basis of fluorescent reporter signals or adult fish on the basis of fin clip junction PCR. During the construction of the Drosophila, or adapted for medical research, including the establishment of disease models for drug screening and mechanism dissection or the tracing of cancer cell transformation and metastasis to provide new insights into carcinogenesis and cancer therapy.Compared with traditional KI strategies, which generally adopt donors containing a single functional cassette, our dual-cassette donor design provides more flexibility to achieve complex genome manipulation, enabling a variety of applications. In addition to coupling conditional knockout with gene-labeling or geno-tagging, this dual-cassette donor system could theoretically be expanded to other applications after further modification , such asFRT, attB/attP, or Dre/Rox to flank the unwanted backbone and then delete it after successful knock in Nevertheless, our current dual-functional donor KI strategy has certain limitations and is open to further improvement. To simplify donor design and construction and reduce the size of the donor plasmids as much as possible, we have omitted most intron sequences downstream of the Cas9/gRNA target sites as well as the endogenous 3’-UTRs, and mainly relied on the downstream coding sequence from the cDNA to rescue the correct expression of the target gene. This approach may alter the expression pattern and/or regulation of the target gene, so we suggest careful evaluation of the reporter gene expression and comparison of the result with the endogenous gene characteristics before performing further experiments, such as conditional knockout or lineage tracing. Of course, one can easily include introns and 3’-UTRs in the donor design when necessary. On the other hand, since only one form of the downstream intronless cDNA sequence is inserted into the final KI allele to complement the disruption of endogenous genes, this approach may not be applicable to genes with multiple alternative splicing isoforms or genes regulated by RNA splicing. However, this limitation may be avoided by including all the downstream introns in the Po-cassette of the donor. In addition, this limitation sometimes might be considered a unique advantage, through which KI alleles with different alternative splicing isoforms could be purposely designed and generated separately, facilitating specific and detailed investigation as well as the comparison of different isoforms. In addition to the disadvantages in the design of the rescue sequences in the Po-cassette, another concern is that all the current KI alleles retain the plasmid backbone sequence at the insertion site in the genome, which might induce DNA methylation and silencing of transcription, as previously reported . In the All the zebrafish used in this study were raised in groups and maintained at 28.5°C in the zebrafish facility of Peking University with a 14 hr/10 hr light/dark cycle. The wild-type strain used was Tübingen (TU). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University. The reference from IACUC of Peking University is LSC-ZhangB-2.tbx5a locus targeting the second intron  as an example, the hEMX1 target site and the first loxP site were linked by a 111 bp DNA fragment cloned from the pMD18-T vector  to avoid the disruption of the loxP sequence due to NHEJ-mediated DSB repair induced by the cleavage of the hEMX1 sequence, and this 1sthEMX1-linker-loxP sequence was cloned into the pMD19-T simple vector . Then, the intron two sequence downstream of the tbx5a I2 target site (including the splice acceptor) of the tbx5a gene was cloned downstream of this first loxP site. The tbx5a partial CDS , BGH PA and a modified exon 3 of tbx5a that contained an in-frame stop codon and included its upstream splice acceptor sequence, were successively cloned downstream of the tdTomato coding sequence. The tbx5a-T2A-tdTomato floxP 2PA-mutExon donor (abbreviated as tbx5a PoR-Ne donor) was used as the basic vector to construct the PoNe donor for the kctd10 gene and for the geno-tagging donors for the tbx5a and sox10 loci. To construct the tbx5a geno-tagging PoR-NeG donor as an example, we improved the initial tbx5a PoR-Ne donor by cloning the sequence around the splice acceptor of the second intron of the tbx5a gene and fused it in-frame with a tdGFP coding sequence, which was separated by a 2A peptide sequence, and we subsequently inserted the whole sequence between the second loxP site and the 2PA; then, another copy of 2PA was inserted upstream of the second loxP site, and the mutExon was removed (To construct the PoNe donor for the  removed .http://casot.cbi.pku.edu.cn/) (5’-AAAAAAAGCACCGACTCGGTGCCAC-3’). Then, gRNAs were synthesized by in vitro transcription with T7 RNA polymerase (TAKARA) and purified by LiCl or ethanol precipitation. The gRNA target sequences are shown in zCas9 mRNA through in vitro transcription using the mMessage mMachine T7 kit (Ambion) (zCas9 mRNA was purified by LiCl precipitation. The Cre expression vector pX-T7-Cre was linearized with XbaI digestion and used as the template for synthesizing Cre mRNA through in vitro transcription by using the mMessage mMachine T7 kit (Ambion). Then, the Cre mRNA was purified by LiCl precipitation.The gRNAs were designed with the CasOT program (edu.cn/) . Forwardedu.cn/) , togethe(Ambion) . Then, tzCas9 mRNA and gRNAs were coinjected into one-cell-stage zebrafish embryos with or without donor plasmids. Each embryo was injected with 1–2 nL of a solution containing 700 ng/μL zCas9 mRNA, 100 ng/μL gRNA and 15 ng/μL donor plasmid; 100 pg or 25 pg of Cre mRNA was injected into 1 cell stage or a single cell of 4 cell stage zebrafish embryos, respectively.The zCas9 mRNA and the corresponding gRNA. To extract genomic DNA, at 24 hpf, fifteen injected embryos were divided into three groups and lysed with 50 mM NaOH solution (10 μL each embryo) at 95°C for 15 min, followed by neutralization with 1 M Tris-HCl . Then, 1 μL of the genomic DNA extract was used as the template to amplify the target site regions with the corresponding primers  or cloned into pMD18-T (TAKARA) for clonal sequencing (F0 embryos).Genomic DNA was extracted with lysis buffer  from either individual or pools of 72 hpf zebrafish embryos and then used to PCR amplify the 5’ and 3’ junction fragments of target genes or the region flanking the  primers . The PCRT2-T2A-ECFP)Tg promoter sequence and the SV40 polyadenylation site were cloned and ligated into the pminiTol2 vector  from 5 hpf to 48 hpf and the embryo medium was changed once at 24 hpf.To construct the plasmid for the generation of 2 vector  by the G CreERT2  and ECFPFor general imaging, zebrafish embryos were anesthetized with 0.02% tricaine , then placed in 3% methylcellulose (Sigma), and imaged under a microscope  equipped with AxioCam MRm (Zeiss). Confocal imaging was performed using an LSM 710 confocal microscope (Zeiss) with a 20X water immersion objective. Z-stack images were acquired with a 5- to 7 μm resolution. The images were processed with AxioVision Rel.4.8 and ZEN 2009 imaging software.Cre mRNA. All the embryos were collected from crosses of the zebrafish carrying the corresponding homozygous KI alleles with either a wild-type or a heterozygote zebrafish. Embryos of different genotypes were distinguished and grouped by fluorescent signals and corresponding phenotypes. At least 500 ng of total RNA was employed for reverse transcription using the 5x All-In-One RT MasterMix . All reactions were performed in at least technical triplicates, and the results represent biological triplicates. Each primer pair was designed to span the KI donor integration site, and gapdh was used as the internal reference gene. qRT-PCR primer information is listed in −ΔΔCt method, and p<0.05 was accepted as statistically significant.RNA was isolated using TRIzol reagent (Invitrogen) according to the standard protocol from 10 randomly picked 72 hpf sibling embryos with or without the injection of  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Acceptance summary:Your manuscript represents a very important technical advance in the emerging field of conditional mutagenesis in the zebrafish. The mutagenesis vectors you have developed offer a key advantage over existing tools: the ability to track homozygous wild type, heterozygous mutant or homozygous mutant status of every cell using fluorescent reporters. Together with well-documented amenability of zebrafish embryos and larvae to high-resolution imaging, these tools should enable tremendous advances in genetic analysis of developmental processes. Furthermore, the ability to unequivocally assign mutant, heterozygous or wild type genotypes to every cell in a mosaic tissue should be of high interest to those studying genetic control of post-embryonic processes such as regeneration. The apparent ease with which your vectors can be integrated into the genome (using non-homologous end joining instead of less efficient but more precise homology directed repair) should make the methodology readily accessible to a large number of laboratories.Decision letter after peer review:eLife. Your article has been reviewed by Didier Stainier as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: David Grunwald (Reviewer #3).Thank you for submitting your article "One-step efficient generation of dual-function conditional knockout and geno-tagging allele in zebrafish" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:loxP sites for Cre-mediated excision. The second part of the integrated transgene contains transcriptional terminators and splice acceptors for mutagenesis using the gene trap principle. Authors demonstrate efficient transgene integration and Cre-dependent mutagenesis of tbx5a, sox10 and kcdt10 genes.The manuscript by Zhang and colleagues describes a novel conditional mutagenesis system for the zebrafish. The system uses non-homologous end joining pathway to integrate dual function transgenes into the genome with very high efficiency. The "rescue" component of the integrated transgene contains the cDNA coding for exons downstream of the integration site co-translationally linked to a fluorescent reporter. The "rescue" cassette is flanked by eLife , is that downstream exons of the mutated gene are not expressed, which should help avoid genetic compensation.While several recent papers have described conditional mutagenesis methods for zebrafish, they have not yet been broadly employed by other laboratories. The approach described in this manuscript is made particularly attractive by the ability to pre-screen using fluorescence, resulting in very high apparent transgenesis rates. The ability to use fluorescence to track mutant and wild type alleles built into the PoR-NeG vectors may be of particular interest for mosaic analysis. An additional advantage, shared with another method recently published in In summary, the manuscript by Zhang et al. represents an important step developing a highly sought-after technique of conditional mutagenesis for the zebrafish.Essential revisions:mutExon in the PoNe donor may induce NMD and genetic compensation if expressed). This compelling hypothesis can only be tested by mutating a gene known to be subject to genetic compensation. To our knowledge, tbx5a, sox10 and kcdt10 loci selected for mutagenesis are not subject to genetic compensation, making them unsuitable for testing the non-compensation hypothesis. Thus, authors must either provide data demonstrating lack of genetic compensation or text sentences describing genetic compensation to the Discussion section, clearly indicating speculative nature of such statements. Furthermore, the possibility that mutExon in PoNe donor may induce compensation needs to be discussed.1) The described method of conditional mutagenesis is very likely to be refractive to genetic compensation (with the caveat that the 2) Expression of insertionally mutated loci has to be quantified in "on" and "off" states. It can be done by qRT-PCR using primers in exons upstream and downstream of the integration site, as is common for gene traps. The need to analyze expression in the "on" state is justified because the gene is expressed from intron-less cDNA with a non-native 3' UTR. It is important to analyze expression in the "off" state because all the coding sequences are present in the genome and mutagenicity relies on the efficiency of splicing and transcriptional termination modules within the inserted transgene.3) A thorough breakdown of transgenesis efficiency must be provided, starting with numbers of injected embryos (if available), embryos screened for fluorescence, percentage/numbers of embryos selected for raising and so forth.mutExon not retained PoR-NeG vectors, why different transcriptional terminators are used in PoR-NeG vectors integrated into tbx5a and sox10 loci? Stability of constructs and transgenes containing two 8X terminators should be discussed. In simplest terms, which vector would you recommend for other labs to use?4) A better explanation for different components used in different vectors must be provided. Specifically, why was 5) Since rescue function is provided by partially intronless cDNA, this approach may not be applicable to genes with multiple alternatively spliced isoforms or genes regulated at the level of RNA splicing. This limitation should be discussed.6) Higher resolution images of transgenes must be included for all figures, especially Figure 4—figure supplement 1.Reviewer #1:loxP sites to allow Cre-dependent removal. The second unit confers transcriptional termination. In the basic vector, it consists of two or more pA signals (SV40 pA and BGH pA) followed by a mutant native exon with a premature stop codon. In the more advanced version, the second unit consists of a traditional gene-trap that could lead to the expression of a different fluorescent protein. The authors inserted the cassettes at 3 different loci  and demonstrated germline transmission in 5-50% founders. They showed the conditionality of these alleles and the utility of alleles generated by the advanced cassette in revealing the genotypes of individual cells. The results are of good quality and the writing is fair. Efficient generation of conditional alleles is a sought-after technique in the zebrafish field. The manuscript therefore addresses an unmet need and should be of interest to scientists in the zebrafish community and beyond. However, there are several issues that need to be addressed, both in the experiments and in the writing.The manuscript by Li et al., "One-step efficient generation of dual-function conditional knockout and geno-tagging allele in zebrafish", describes the authors successful attempts in generating dual functional alleles in zebrafish using NHEJ-mediated insertion targeted by Cas9/gRNA. This gene-trap approach uses two functional units in the vectors. The first unit confers cis-complementation of the mutagenic effect and expression of a fluorescent protein. It consists of the native splice acceptor, the remaining downstream coding sequence, and the coding sequence of a fluorescent protein. The unit is flanked by Essential revisions:1) There is no quantitative evaluation of the effects of the KI alleles on gene expression. The authors only qualitatively assessed the KI alleles by comparing the expression patterns of the fluorescent protein(s) and the major phenotypes of the homozygous mutants. Quantitative evaluation of such alleles in "normal" and "defective" state at the mRNA level is a norm and should be done here. It is important to know how normal is "normal" and how defective is "defective".2PA is highly effective as the transcriptional terminator, they replaced it with 8PA in the last cassette. The stability of two tandem 8PA needs to be evaluated.2) The cassette design seems random, not systematic. The authors used 3 different designs. They discussed potential benefits of each feature but often without strong experimental evidence. When adding more features to the more advanced cassettes, they also dropped certain presumably important feature without rationale. For example, the authors argument that the mutant exon with premature termination codon in the cassette may be indispensable for effective gene disruption, but there is no direct evidence. They then dropped the mutant exon in the more advanced cassettes. Although the authors argued that 3) The authors argued that one of the main advantages of the described alleles is that they may bypass transcriptional adaptation elicited by a premature termination codon. Yet they included a premature termination codon in their cassette.4) All the alleles also retain the backbone sequence of the vector at the insert site. It has been reported that such extraneous sequence can induce DNA methylation and silencing. This needs to be discussed.5) The resultant "normal" alleles have reduced intron number, which may alter the expression, or eliminate certain alternative spliced forms. This needs to be discussed as potential disadvantage.emx1 gRNA. It is unclear whether the lamGolden gRNA also requires LiCl precipitation for activity.6) The necessity of LiCl precipitation of gRNA is confusing. Although the observation is intriguing, but it is only true for the Reviewer #2:tbx5a, kctd10 and sox10). In each case they developed reporter alleles that were tagged with fluorescent proteins and could be inactivated by Cre mRNA injection.In the present report by Li et al. the authors describe a genetic method to generate conditional knock out alleles in zebrafish based on the Cre/Lox system and CRISPR/CAS9 mediated NHEJ. With this technique the authors generate specific alleles in few target loci .In addition, it is important to test their floxed alleles, crossing them with Cre expressing lines in specific tissues (for instance the heart primordium) to show that tissue and temporal control of the recombination event can be achieved.Without these experiments the paper shows only a very limited advancement in designing clever targeting plasmid but fails to proof the real power of this approach.Reviewer #3:Li et al. introduce a new method for creating conditional knockout alleles in the zebrafish. The method generates a wildtype allele translationally linked to a fluorescent protein so that cells expressing the wildtype allele can be identified. Cre activity leads to excision of the wildtype-fluorescent reporter sequences and leads to expression of a different reporter protein so that the loss-of-function allele is tagged. It is a very nice idea and it will be very useful. The method is not perfect in that the wildtype activity is supplied by a cDNA sequence and the claim is that the mutant allele will not trigger transcription compensation, which is not tested and may or may not be true, but this method is a true advance in the field and will be implemented by others. The method deserves to be published.Overall the manuscript is very well written. Nevertheless, the manuscript has a number of areas that require clarification. At present, a number of the experiments presented in the figures are difficult to interpret. The paper will make a much bigger impact if the reader has an easy time understanding the experiments and the results.must be corrected. The authors claim throughout the paper that they have improved the efficiency of insertion or of generating insertions that will be transmissible through the germline. This is not correct. They have created a method that allows for the efficient recovery of germline transmissible modified alleles. They have made a wonderful advance but it is not the same as improving recombination rates. The authors have come up with a wonderful method for pre-selecting embryos that have acquired insertions in somatic tissue, because integration in the correct orientation leads to expression of a fluorescent reporter under control of the targeted gene. A low percentage of injected embryos grow into normal-looking embryos with reporter expression . Then among those animals pre-selected for the presence integration events, a reasonable and varied fraction will transmit the edited allele to the next generation. Therefore, it is simply false for the authors to write "The feasibility of this strategy was demonstrated at tbx5a and kctd1 loci, with germline transmission efficiency as high as 56%." Such a statement would be interpreted by readers as indicating that they had greatly advanced targeting efficiency so that 56% of injected animals will transmit an edited allele. Similarly, in the Discussion, the authors claim: "As high as 50% of the F0 fish could transmit the integrated donor construct to their offspring, which is much higher in germline transmission efficiency than previously reported HR-mediated gene knock-in." Instead, the authors should say they have developed a clever method that makes recovery of conditional alleles very efficient. This method involves first preselecting embryos that have mosaically acquired the conditional allele, and then only screening the preselected transgenic animals for the ability to transmit the alleles through the germline. Under these conditions, up to 56% of the pre-selected founders may transmit edited alleles. The authors should make this clear in the Abstract also.1) The authors do not correctly describe the efficiency rates of generating insertions or the efficiency with which germline insertions are made or recovered. This is a very important point and tbx5a locus". The authors write: "After injection and screening, integration of this donor and germline transmission of the tbx5a PoR-NeG donor geno-tagging alleles were detected and confirmed in 2 out of 48 adult F0". I can't understand where the number "48" comes from – was this all the F0 adults from an injection? Was it 48 F0 selected on the basis of fluorescent reporter expression? In Table 2, row 3 it appears that 16/124 injected embryos had reporter expression – so where did the 48 come from?2) It is difficult to reconstruct and to figure out the actual numbers that describe the recovery of germline transmissible alleles. For example, let's look at the section that begins subsection "Generation and evaluation of geno-tagging alleles at zebrafish cmcl2:EGFP). As a result, if the WT allele were clearly marked as PoR-Ne then it would be easy to tell which is the heart marker and which is the tagged gene. In general: please use consistent nomenclature.3) The authors create conditional alleles that can be switched from WT red to mutant green. They call these PoR-NeG alleles for Positive Red to Negative Green. They also seem to call these geno-tagged alleles. They sometimes breed these to animals with another type of condition allele that switches from a WT-fluorescent reporter to a mutant-no reporter allele. They call these alleles PoNe, positive to negative. It would make the reading much easier if they always labeled an allele that co-expressed a fluorescent reporter. In other words, when the wildtype allele is marked by expression of tdTomato and the mutant allele is not marked by reporter expression, maybe call it PoR-Ne. In addition, they place these PoNe alleles on backgrounds that express a heart reporter (4) The figures need some work. The easiest suggestion is that the lettering needs to be larger or at higher resolution – especially for the line drawings – when I expanded it on my screen it became very pixelated. For example, try enlarging Figure 4—figure supplement 1F.5) Figure 2A and C: I find these very hard to interpret – I think the authors are visualizing fluorescent markers in three embryo siblings generated from a single cross some of which have been injected with Cre mRNA. The patterns of fluorescence reporter expression differ and I believe the results are to be interpreted as being derived from embryos with different genotypes. Would it be possible for the authors to tell us the presumed genotypes of each row before Cre exposure? I feel this would make the interpretation easier for the reader. I don't understand Figure 2C – it looks like each embryo has a green heart but I think the authors want us to focus on differences. These need to be spelled out perhaps in the figure legend? For Panel E please explain in the figure legend this experiment. Explain the cross and what Normal and Defective embryos are. Explain what "before injection" means.6) Figure 3B: I suspect the markers on the gel are mistakenly labeled. For example, the experimental band of 380 bp is larger than the marker of 500 bp and the experimental band of 820 bp is smaller than the marker of 750 bp. Also, in Panel B, the gel of the 3' junctions: I don't understand how amplification from the donor shown in Panel A with T5F2 and T5R1 would give a band – is there some mistake here?7) In Figure 3C, I believe the images of dorsal view and ventral view have been reversed. I think the first column and bottom row picture is a ventral view. Please check – I may be wrong. I think the cross that produced the embryos in Panel C needs to be clearly explained and how to interpret the genotypes should be clearly explained. The images are difficult to interpret – what are we supposed to look at in the boxed insets? Explain in the legend please.8) The images in Figure 3—figure supplement 1 are difficult to interpret. Again I suspect that if the authors told us the predicted genotypes and told us what we are supposed to be seeing in the outlined boxed areas, then it would be clearer.kctd10 locus”: it the sentence beginning “Interestingly, in the initial design of the kctd10 PoNe donor…” might be easier to read if you re-phrased this sentence something like: "In initial experiments, we used only a donor with a single SV40pA termination signal sequence without the modified exon (mutExon). With such donors we were able to isolate stable integrations at the kctd10 locus and observed the correct expression pattern of tdGFP.”9) Subsection “Generation and evaluation of dual-function alleles showing CKO with gene labeling effect at zebrafish eLife. Your revised article has been favorably evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "One-step efficient generation of dual-function conditional knockout and geno-tagging allele in zebrafish" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:eLife paper describing a conditional knock-in allele. This is very important. As one can appreciate from error bars in Figure 2—figure supplement 1E, Figure 2—figure supplement 2L, Figure 3—figure supplement 1E and Figure 4—figure supplement 1G, it is impossible to distinguish between 90% and 99% mutagenesis efficiency in heterozygotes (55% vs. 51% of read-through transcript remaining in heterozygotes). In contrast, the difference between 10% and 1% levels of remaining wild-type transcript should be quite straightforward to reliably quantify in homozygotes. Results of such quantitative analysis may have an impact on the choice between your described polyA cassettes (tandem SV40/GBH vs. 8xSV40). Thus, qRT-PCR analyses must be performed on embryos homozygous for Cre-excised alleles.1) The revised manuscript is much improved, and additional data demonstrating feasibility of fully conditional knockout using the new cmlc2:CreERT2 line. However, one major issue remains. It is common practice to measure the expression of targeted loci in the "off" state in homozygotes. Data can be displayed in several different ways, exemplified by Figure 2 in Ni et al., 2012 reference and Figure 1D in the Grajevskaja et al., 2018. Figure 1A in Sugimoto et al., 2017 serves as a directly relevant example of from a recently published 2) A second issue somewhat detracting from the overall quality of the work is occasionally difficult-to-read English. I would simply recommend that the final submission should be edited with the help of a professional editor.3) Sequences of primers used for qRT-PCR should be included, along with other primers, in Supplementary file 7.kctd10 deleted from the Abstract?4) Why is tbx5a locus”: Need to show hEMX1 target site on the vector in Figure 1A, and corresponding sites in vector diagrams in subsequent figures.5) Subsection “High efficient generation of a dual-function KI allele at zebrafish  Essential revisions:1) The described method of conditional mutagenesis is very likely to be refractive to genetic compensation (with the caveat that the mutExon in the PoNe donor may induce NMD and genetic compensation if expressed). This compelling hypothesis can only be tested by mutating a gene known to be subject to genetic compensation. To our knowledge, tbx5a, sox10 and kcdt10 loci selected for mutagenesis are not subject to genetic compensation, making them unsuitable for testing the non-compensation hypothesis. Thus, authors must either provide data demonstrating lack of genetic compensation or text sentences describing genetic compensation to the Discussion section, clearly indicating speculative nature of such statements. Furthermore, the possibility that mutExon in PoNe donor may induce compensation needs to be discussed.egfl7 locus, to evaluate the possibility of eliminating genetic compensation by our method. Unfortunately, we have not identified satisfactory high-efficient Cas9/gRNA target sites in the introns of this gene so far, preventing us from generating KI lines for the evaluation. We have modified our statements about genetic compensation following your suggestions, including indicating speculative nature of the non-compensation claims as well as the possibility that mutExon in the PoNe donor may induce compensation, and moved the corresponding sentences to Discussion section.Sorry for the confusion in our statements about genetic compensation. We have tried to generate CKO+gene-labeling and geno-tagging alleles for the 2) Expression of insertionally mutated loci has to be quantified in "on" and "off" states. It can be done by qRT-PCR using primers in exons upstream and downstream of the integration site, as is common for gene traps. The need to analyze expression in the "on" state is justified because the gene is expressed from intron-less cDNA with a non-native 3' UTR. It is important to analyze expression in the "off" state because all the coding sequences are present in the genome and mutagenicity relies on the efficiency of splicing and transcriptional termination modules within the inserted transgene.tbx5a, kctd10 and sox10 loci, in 72 hpf embryos obtained from outcross of respective KI homozygous fish with a wild-type one, with or without Cre mRNA injection. As an example , the results from the tbx5a PoR-Ne donor KI fish showed that the mRNA transcription level of the tbx5a locus in the +/PoR-Netbx5a embryos before Cre mRNA injection was comparable with that of wild-type control embryos, indicating normal expression of the PoR-Netbx5a allele . After Cre mRNA injection, no red fluorescence signal could be detected in the +/PoR-Netbx5a embryos, indicating efficient excision of the Po-cassette and most of the PoR-Netbx5a alleles had been efficiently converted into Netbx5a alleles. The tbx5a expression level in these +/Netbx5a embryos significantly decreased to around 50% of the level in the wild-type embryos, as expected . Similar results were obtained for the cross from either tbx5a or sox10 PoR-NeG geno-tagging fish , or kctd10 PoG-Ne KI fish .Thank you for the suggestion. We have designed primers flanking the insertion sites and performed qRT-PCR experiments to quantify the “on” and “off” states of the PoNe and geno-tagging alleles of 3) A thorough breakdown of transgenesis efficiency must be provided, starting with numbers of injected embryos (if available), embryos screened for fluorescence, percentage/numbers of embryos selected for raising and so forth.Sorry for the incompleteness of these important details about the injection and screening process. We have provided more detailed information about these process in the revised manuscript, as the following:tbx5a KI allele”: “To generate tbx5a PoR-Ne KI zebrafish, about 100 one-cell stage embryos were injected, and red fluorescent signals could be detected in 18 of them (Supplementary file 2). […] In contrast, no red fluorescent signal was detected in any F1 progeny from outcross of 40 adult F0 raised from tdTomato-negative embryos, indicating a positive correlation of reporter gene expression in founder embryos with successful as well as efficient germline transmission of the KI events.”1) An extract from subsection “Germline transmission and evaluation of the kctd10 locus”: “For the generation of kctd10 PoG-Ne KI fish line, we injected about 100 one-cell stage embryos and observed the mosaic expression of tdGFP in 22 F0 embryos (Supplementary file 2). We raised all the F0 embryos without pre-selection to adulthood, and found 2 of 42 (4.8%) displayed germline transmission screening of 42 F0 adults, only two of them displayed germline transmission of the kctd10 PoG-Ne donor KI events (Supplementary file 4).”2) An extract from subsection “Generation and evaluation of dual-function alleles showing CKO with gene labeling effect at zebrafish tbx5a locus”: “To generate tbx5a PoR-NeG geno-tagging zebrafish, about 100 one-cell stage embryos were injected, and 16 of them showed mosaic expression of red fluorescent signals (Supplementary file 2), whose ratio is comparable with the previous tbx5a PoR-Ne donor KI experiment. […] These results indicate a strong correlation between germline transmission of KI events and adult somatic tissue integration of the donor, and provide another efficient method for the enrichment of germline transmission events.”3) An extract from subsection “Generation and evaluation of geno-tagging alleles at zebrafish sox10 gene”: “To generate the heritable sox10 PoR-NeG geno-tagging allele, about 150 one-cell stage embryos were injected with the KI system (without Cre mRNA), and the tdTomato fluorescent signals were observed in 103 F0 embryos (Supplementary file 2). The positive founder embryos showing fluorescent signals were raised to adulthood and germline transmission screening was conducted by outcross with wild-type zebrafish. Correct red fluorescent patterns in F1 embryos were observed in 10 out of 21 founders (Supplementary file 6).”4) An extract from subsection “Geno-tagging effect achieved for zebrafish In addition, we added a new paragraph in the Discussion section and summarized as well as further discussed the striking correlation between efficient germline transmission and correct insertions of the KI donors in somatic tissues.4) A better explanation for different components used in different vectors must be provided. Specifically, why was mutExon not retained PoR-NeG vectors, why different transcriptional terminators are used in PoR-NeG vectors integrated into tbx5a and sox10 loci? Stability of constructs and transgenes containing two 8X terminators should be discussed. In simplest terms, which vector would you recommend for other labs to use?mutExon (including its preceding splice acceptor sequence) bearing a premature stop codon was included in the Ne-cassette, following the 2X PA terminators, of the initial version of the PoNe donor, where only a single fluorescent reporter gene (tdTomato or tdGFP) was incorporated into the Po-cassette. In case there is leaky transcription after the 2X PA, this mutExon will be transcribed and spliced into the mature mRNA, ensuring disruption of the function of the target gene. This type of donor was used to generate the PoR-Ne, kctd10PoG-Ne-1tbx5a and PoG-Ne-2kctd10 alleles. However, since the presence of mutExon will disrupt the translation of the fluorescent reporter in the Ne-cassette (of the PoR-NeG donor), and on the other hand, it might trigger genetic compensation, in the design of geno-tagging PoR-NeG donor, the advanced version of the original PoNe donor, we replaced the mutExon with its wild-type counterpart, to ensure normal expression of the fluorescent reporter in the Ne-cassette after Cre-induced removal of the Po-cassette, as well as to bypass the unwanted genetic compensation response.Sorry for the confusion. The key to the success of our dual-function KI strategy relies on the special design of the dual-cassette donor. In order to refine our method, we have tried slightly different designs on certain components in different KI experiments, on the premise of keeping the Po- and Ne-cassettes intact and functional. Since polyA signals are generally not 100% efficient for the termination of transcription, a mutExon for the design of PoR-NeG geno-tagging donor, another concern is the efficiency of transcriptional termination of the Po-cassette, since its leaky transcription might lead to unwanted expression of the fluorescent reporter in the Ne-cassette before Cre-induction. It has been reported that the efficiency of transcriptional termination correlates with the number of polyA signals . Although we have obtained satisfactory results with the original 2X PA for the generation of PoR-NeGtbx5a geno-tagging allele, we decided to try more polyA signals for the sox10 locus, concerning that the expression of sox10 is more ubiquitous and seems also stronger than that of tbx5a. Therefore, to be on the safe side, we expanded the original single SV40 polyA signal into eight tandem repeats (8xPA) for stronger transcriptional termination in the Po-cassette, and also used this 8X PA sequence for the transcriptional termination of the Ne-cassette. Indeed, there are potential disadvantages for the employment of two 8X PA sequences, each containing eight times tandem repeats of the ~160-bp polyA signal sequence, in the sox10 PoR-NeG geno-tagging donor as well as the PoR-NeGsox10 allele, when concerning the stability. On one hand, the repeats within each 8X PA might be reduced during expansions and/or passages to next generations of this allele; On the other hand, the sequence flanked by the two 8X PA (containing the downstream loxP and the Ne-cassette) might be lost due to occasional recombination of the two 8X PA, leading to abolishment of the CKO and fluorescence-switch effect. Unfortunately, at present we have’t been able to assess the situation of these 8X PA sequences in the transgenic fish by PCR amplification and sequencing, due to its highly repetitive nature. However, so far, the PoR-NeGsox10 allele behaves normally, considering the observation that the F2 embryos displayed expected expression of tdTomato signal under normal condtions and also showed expected switch to tdGFP expression upon treatment with Cre recombinase, indicating that the two 8X PA sequences are still functional. In the future, we will keep tracking the inheritance and stability of this allele, and also will try to establish a method to evaluate the situation of the 8X PA sequences in the transgenic fish.In addition to avoiding the PA, and then testing its effect in founder embryos to see whether it is enough to stop the expression of the second fluorescent reporter, and introducing more polyA signals when necessary.Taking into account of all the above concerns for the donor design, we recommend to use the geno-tagging donor for the generation of CKO allele coupled with gene or allele labeling effect, beginning with constructing the donor with 2X We have clarified this issue by including the above explanations and discussions in the sections of both Results section and Discussion section in our revised manuscript.5) Since rescue function is provided by partially intronless cDNA, this approach may not be applicable to genes with multiple alternatively spliced isoforms or genes regulated at the level of RNA splicing. This limitation should be discussed.Thank you for reminding us the limitations of our method. We have clarified this issue by adding a new paragraph in the Discussion section, discussed the limitations of our current strategy, including the concerns about splicing regulation, and also gave some suggestions of potential solutions to these problems.6) Higher resolution images of transgenes must be included for all figures, especially Figure 4—figure supplement 1.Sorry for the quality of the figures in the manuscript of our initial submission. Now we have found a better method to preserve high resolution of our figures during conversion into the PDF file, and we believe that the new figures are satisfactory in quality and could display clear details after enlargement.Reviewer #1:1) There is no quantitative evaluation of the effects of the KI alleles on gene expression. The authors only qualitatively assessed the KI alleles by comparing the expression patterns of the fluorescent protein(s) and the major phenotypes of the homozygous mutants. Quantitative evaluation of such alleles in "normal" and "defective" state at the mRNA level is a norm and should be done here. It is important to know how normal is "normal" and how defective is "defective".tbx5a, kctd10 and sox10 loci, in 72 hpf embryos obtained from outcross of respective KI homozygous fish with a wild-type one, with or without Cre mRNA injection. As an example , the results from the tbx5a PoR-Ne donor KI fish showed that the mRNA transcription level of the tbx5a locus in the +/PoR-Netbx5a embryos before Cre mRNA injection was comparable with that of wild-type control embryos, indicating normal expression of the PoR-Netbx5a allele . After Cre mRNA injection, no red fluorescence signal could be detected in the +/PoR-Netbx5a embryos, indicating efficient excision of the Po-cassette and most of the PoR-Netbx5a alleles had been efficiently converted into Netbx5a alleles. The tbx5a expression level in these +/Netbx5a embryos significantly decreased to around 50% of the level in the wild-type embryos, as expected . Similar results were obtained for the cross from either tbx5a or sox10 PoR-NeG geno-tagging fish , or kctd10 PoG-Ne KI fish .Thank you for the suggestion. We have designed primers flanking the insertion sites and performed qRT-PCR experiments to quantify the “normal” and “defective” states of the PoNe and geno-tagging alleles of 2) The cassette design seems random, not systematic. The authors used 3 different designs. They discussed potential benefits of each feature but often without strong experimental evidence. When adding more features to the more advanced cassettes, they also dropped certain presumably important feature without rationale. For example, the authors argument that the mutant exon with premature termination codon in the cassette may be indispensable for effective gene disruption, but there is no direct evidence. They then dropped the mutant exon in the more advanced cassettes. Although the authors argued that 2PA is highly effective as the transcriptional terminator, they replaced it with 8PA in the last cassette. The stability of two tandem 8PA needs to be evaluated.mutExon (including its preceding splice acceptor sequence) bearing a premature stop codon was included in the Ne-cassette, following the 2X PA terminators, of the initial version of the PoNe donor, where only a single fluorescent reporter gene (tdTomato or tdGFP) was incorporated into the Po-cassette. In case there is leaky transcription after the 2X PA, this mutExon will be transcribed and spliced into the mature mRNA, ensuring disruption of the function of the target gene. This type of donor was used to generate the PoR-Ne, kctd10PoG-Ne-1tbx5a and PoG-Ne-2kctd10 alleles. However, since the presence of mutExon will disrupt the translation of the fluorescent reporter in the Ne-cassette (of the PoR-NeG donor), and on the other hand, it might trigger genetic compensation, in the design of geno-tagging PoR-NeG donor, the advanced version of the original PoNe donor, we replaced the mutExon with its wild-type counterpart, to ensure normal expression of the fluorescent reporter in the Ne-cassette after Cre-induced removal of the Po-cassette, as well as to bypass the unwanted genetic compensation response.Sorry for the confusion. The key to the success of our dual-function KI strategy relies on the special design of the dual-cassette donor. In order to refine our method, we have tried slightly different designs on certain components in different KI experiments, on the premise of keeping the Po- and Ne-cassettes intact and functional. Since polyA signals are generally not 100% efficient for the termination of transcription, a mutExon for the design of PoR-NeG geno-tagging donor, another concern is the efficiency of transcriptional termination of the Po-cassette, since its leaky transcription might lead to unwanted expression of the fluorescent reporter in the Ne-cassette before Cre-induction. It has been reported that the efficiency of transcriptional termination correlates with the number of polyA signals . Although we have obtained satisfactory results with the original 2X PA for the generation of PoR-NeGtbx5a geno-tagging allele, we decided to try more polyA signals for the sox10 locus, concerning that the expression of sox10 is more ubiquitous and seems also stronger than that of tbx5a. Therefore, to be on the safe side, we expanded the original single SV40 polyA signal into eight tandem repeats (8xPA) for stronger transcriptional termination in the Po-cassette, and also used this 8X PA sequence for the transcriptional termination of the Ne-cassette. Indeed, there are potential disadvantages for the employment of two 8X PA sequences, each containing eight times tandem repeats of the ~160-bp polyA signal sequence, in the sox10 PoR-NeG geno-tagging donor as well as the PoR-NeGsox10 allele, when concerning the stability. On one hand, the repeats within each 8X PA might be reduced during expansions and/or passages to next generations of this allele; On the other hand, the sequence flanked by the two 8X PA (containing the downstream loxP and the Ne-cassette) might be lost due to occasional recombination of the two 8X PA, leading to abolishment of the CKO and fluorescence-switch effect. Unfortunately, at present we have’t been able to assess the situation of these 8X PA sequences in the transgenic fish by PCR amplification and sequencing, due to its highly repetitive nature. However, so far the PoR-NeGsox10 allele behaves normally, considering the observation that the F2 embryos displayed expected expression of tdTomato signal under normal condtions and also showed expected switch to tdGFP expression upon treatment with Cre recombinase, indicating that the two 8X PA sequences are still functional. In the future, we will keep tracking the inheritance and stability of this allele, and also will try to establish a method to evaluate the situation of the 8X PA sequences in the transgenic fish.In addition to avoiding the PA, and then testing its effect in founder embryos to see whether it is enough to stop the expression of the second fluorescent reporter, and introducing more polyA signals when necessary.Taking into account of all the above concerns for the donor design, we recommend to use the geno-tagging donor for the generation of CKO allele coupled with gene or allele labeling effect, beginning with constructing the donor with 2X We have clarified this issue by including the above explanations and discussions in the sections of both Results section and Discussion section in our revised manuscript.3) The authors argued that one of the main advantages of the described alleles is that they may bypass transcriptional adaptation elicited by a premature termination codon. Yet they included a premature termination codon in their cassette.mutExon bearing a premature stop codon in the Ne-cassette of the original single fluorescent reporter version of the PoNe donor, as seen in the PoR-Ne, kctd10PoG-Ne-1tbx5a and PoG-Ne-2kctd10 alleles, which are still prone to genetic compensation or transcriptional adaptation. This is one of the reasons for us to improve the design and came up with the advanced geno-tagging PoR-NeG donor, as represented by the PoR-NeGtbx5a and PoR-NeGsox10 alleles, where the mutExon was replaced by its wild-type counterpart and another fluorescent reporter, which theoretically could be exempted from transcriptional adaptation. We have clarified this issue by adding the corresponding descriptions and discussions in our revised manuscript .Sorry for the confusion. Yes, we indeed included a 4) All the alleles also retain the backbone sequence of the vector at the insert site. It has been reported that such extraneous sequence can induce DNA methylation and silencing. This needs to be discussed.Thank you for pointing out this issue. We have discussed this concern and also provided potential solutions in our revised manuscript (Discussion section).5) The resultant "normal" alleles have reduced intron number, which may alter the expression, or eliminate certain alternative spliced forms. This needs to be discussed as potential disadvantage.sox10 gene”).Thank you for pointing out this potential disadvantage. We have clarified this issue by adding a new paragraph in the Discussion, discussed the limitations of our current strategy, including the concerns about potential alteration of gene expression as well as splicing regulation, and also gave some suggestions of potential solutions to these problems (subsection “Geno-tagging effect achieved for zebrafish 6) The necessity of LiCl precipitation of gRNA is confusing. Although the observation is intriguing, but it is only true for the emx1 gRNA. It is unclear whether the lamGolden gRNA also requires LiCl precipitation for activity.lamGolden gRNA and found similar result as hEMX1 gRNA, where only LiCl precipitation gave satisfactory KI efficiency. We have clarified this issue by adding this information in our revised manuscript (subsection “Geno-tagging effect achieved for zebrafish sox10 gene”).Sorry for the confusion. We have compared the effect of LiCl precipitation with ethanol for the Reviewer #2:In the present report by Li et al. the authors describe a genetic method to generate conditional knock out alleles in zebrafish based on the Cre/Lox system and CRISPR/CAS9 mediated NHEJ. With this technique the authors generate specific alleles in few target loci . In each case they developed reporter alleles that were tagged with fluorescent proteins and could be inactivated by Cre mRNA injection.The use of NHEJ manipulate endogenous loci in zebrafish with high efficiency was previously reported in various papers. In particular a strategy to tag endogenous proteins at the C-terminus in zebrafish was previously reported by Li et al., 2015. The present manuscript is an improvement of the current methods that consists basically in the design of two loxP sites around the artificial tagged exon that is inserted by NHEJ.The authors claim that their strategy will offer novel advantages including the avoidance of genetic compensation mechanisms and the possibility to generate conditional knock out alleles. Although I agree in principle with these statements these are not shown in the current paper.In particular the authors should use their method to silence a known locus that is triggering genetic compensation when inactivated with the insertion of classical indel mutations (as described in the recent literature).In addition, it is important to test their floxed alleles, crossing them with Cre expressing lines in specific tissues (for instance the heart primordium) to show that tissue and temporal control of the recombination event can be achieved.Without these experiments the paper shows only a very limited advancement in designing clever targeting plasmid but fails to proof the real power of this approach.egfl7 locus, to evaluate the possibility of eliminating genetic compensation by our method. Unfortunately, we have not identified satisfactory high-efficient Cas9/gRNA target sites in the introns of this gene so far, preventing us from generating KI lines for the evaluation. We have modified our statements about genetic compensation, indicating speculative nature of the non-compensation claims for our method, and moved the corresponding sentences to Discussion section.Thank you for the suggestions. We have tried to generate CKO+gene-labeling and geno-tagging alleles for the Cre transgenic line Tg(cmlc2:zCreERT2-2A-ECFP) showing expression of both ECFP and 4-hydroxyltamoxifen (4-HT)-inducible CreERT2. This line was crossed with the PoR-NeG/PoR-NeGtbx5a geno-tagging adult fish, and the embryos were treated with 4-HT from 5 hpf to 48 hpf. As expected, green fluorescent signals were clearly detected in the ECFP-positive embryos but not in the un-treated control group, indicating successful switching of tdTomato to tdGFP expression . We have included this new result in our revised manuscript .In order to prove that tissue and temporal control of Cre-induced recombination events can be achieved in our KI transgenic zebrafish, we generated a heart specific Reviewer #3:Overall the manuscript is very well written. Nevertheless, the manuscript has a number of areas that require clarification. At present, a number of the experiments presented in the figures are difficult to interpret. The paper will make a much bigger impact if the reader has an easy time understanding the experiments and the results.1) The authors do not correctly describe the efficiency rates of generating insertions or the efficiency with which germline insertions are made or recovered. This is a very important point and must 0 fish could transmit the integrated donor construct to their offspring, which is much higher in germline transmission efficiency than previously reported HR-mediated gene knock-in." Instead, the authors should say they have developed a clever method that makes recovery of conditional alleles very efficient. This method involves first preselecting embryos that have mosaically acquired the conditional allele, and then only screening the preselected transgenic animals for the ability to transmit the alleles through the germline. Under these conditions, up to 56% of the pre-selected founders may transmit edited alleles. The authors should make this clear in the Abstract also.be corrected. The authors claim throughout the paper that they have improved the efficiency of insertion or of generating insertions that will be transmissible through the germline. This is not correct. They have created a method that allows for the efficient recovery of germline transmissible modified alleles. They have made a wonderful advance but it is not the same as improving recombination rates. The authors have come up with a wonderful method for pre-selecting embryos that have acquired insertions in somatic tissue, because integration in the correct orientation leads to expression of a fluorescent reporter under control of the targeted gene. A low percentage of injected embryos grow into normal-looking embryos with reporter expression . Then among those animals pre-selected for the presence integration events, a reasonable and varied fraction will transmit the edited allele to the next generation. Therefore, it is simply false for the authors to write "The feasibility of this strategy was demonstrated at tbx5a and kctd1 loci, with germline transmission efficiency as high as 56%." Such a statement would be interpreted by readers as indicating that they had greatly advanced targeting efficiency so that 56% of injected animals will transmit an edited allele. Similarly, in the Discussion, the authors claim: "As high as 50% of the Ftbx5a locus” and “Improving the dual-cassette-donor strategy to achieve geno-tagging effect”, and the Discussion section).Sorry for the confusion in the descriptions about the KI efficiency. We have clarified this issue throughout the section of Results and emphasized the importance of pre-selection of founders to achieve successful germline transmission of KI events in the section of Discussion in our revised manuscript  It is difficult to reconstruct and to figure out the actual numbers that describe the recovery of germline transmissible alleles. For example, let's look at the section that begins subsection "Generation and evaluation of geno-tagging alleles at zebrafish tbx5a locus". The authors write: "After injection and screening, integration of this donor and germline transmission of the tbx5a PoR-NeG donor geno-tagging alleles were detected and confirmed in 2 out of 48 adult FSorry for the confusion in the descriptions of our experimental procedures. We have clarified this issue by providing more detailed information about the injection and screening process throughout the section of Results (as summarized in the following) and summarized and discussed the results in the section of Discussion in our revised manuscript. The following is a brief summary:tbx5a KI allele”: “To generate tbx5a PoR-Ne KI zebrafish, about 100 one-cell stage embryos were injected, and red fluorescent signals could be detected in 18 of them (Supplementary file 2). […] In contrast, no red fluorescent signal was detected in any F1 progeny from outcross of 40 adult F0 raised from tdTomato-negative embryos, indicating a positive correlation of reporter gene expression in founder embryos with successful as well as efficient germline transmission of the KI events.”1) An extract from subsection “Germline transmission and evaluation of the kctd10 locus”: “For the generation of kctd10 PoG-Ne KI fish line, we injected about 100 one-cell stage embryos and observed the mosaic expression of tdGFP in 22 F0 embryos (Supplementary file 2). We raised all the F0 embryos without pre-selection to adulthood, and found 2 of 42 (4.8%) displayed germline transmission screening of 42 F0 adults, only two of them displayed germline transmission of the kctd10 PoG-Ne donor KI events (Supplementary file 4).”2) An extract from subsection “Generation and evaluation of dual-function alleles showing CKO with gene labeling effect at zebrafish tbx5a locus”: “To generate tbx5a PoR-NeG geno-tagging zebrafish, about 100 one-cell stage embryos were injected, and 16 of them showed mosaic expression of red fluorescent signals (Supplementary file 2), whose ratio is comparable with the previous tbx5a PoR-Ne donor KI experiment. […] These results indicate a strong correlation between germline transmission of KI events and adult somatic tissue integration of the donor, and provide another efficient method for the enrichment of germline transmission events.”3) An extract from subsection “Generation and evaluation of geno-tagging alleles at zebrafish sox10 gene”: “To generate the heritable sox10 PoR-NeG geno-tagging allele, about 150 one-cell stage embryos were injected with the KI system (without Cre mRNA), and the tdTomato fluorescent signals were observed in 103 F0 embryos (Supplementary file 2). The positive founder embryos showing fluorescent signals were raised to adulthood and germline transmission screening was conducted by outcross with wild-type zebrafish. Correct red fluorescent patterns in F1 embryos were observed in 10 out of 21 founders (Supplementary file 6).”4) An extract from subsection “Geno-tagging effect achieved for zebrafish 3) The authors create conditional alleles that can be switched from WT red to mutant green. They call these PoR-NeG alleles for Positive Red to Negative Green. They also seem to call these geno-tagged alleles. They sometimes breed these to animals with another type of condition allele that switches from a WT-fluorescent reporter to a mutant-no reporter allele. They call these alleles PoNe, positive to negative. It would make the reading much easier if they always labeled an allele that co-expressed a fluorescent reporter. In other words, when the wildtype allele is marked by expression of tdTomato and the mutant allele is not marked by reporter expression, maybe call it PoR-Ne. In addition, they place these PoNe alleles on backgrounds that express a heart reporter (cmcl2:EGFP). As a result, if the WT allele were clearly marked as PoR-Ne then it would be easy to tell which is the heart marker and which is the tagged gene. In general: please use consistent nomenclature.Sorry for the confusion and thank you for the suggestion. We have clarified this issue as suggested in our revised the manuscript.4) The figures need some work. The easiest suggestion is that the lettering needs to be larger or at higher resolution – especially for the line drawings – when I expanded it on my screen it became very pixelated. For example, try enlarging Figure 4—figure supplement 1F.Now we have found a better method to preserve high resolution of our figures, and we believe that the new figures are satisfactory in quality and could display clear details after enlargement.5) Figure 2A and C: I find these very hard to interpret – I think the authors are visualizing fluorescent markers in three embryo siblings generated from a single cross some of which have been injected with Cre mRNA. The patterns of fluorescence reporter expression differ and I believe the results are to be interpreted as being derived from embryos with different genotypes. Would it be possible for the authors to tell us the presumed genotypes of each row before Cre exposure? I feel this would make the interpretation easier for the reader. I don't understand Figure 2C – it looks like each embryo has a green heart but I think the authors want us to focus on differences. These need to be spelled out perhaps in the figure legend? For Panel E please explain in the figure legend this experiment. Explain the cross and what Normal and Defective embryos are. Explain what "before injection" means.Sorry for the confusion and thank you for the suggestion. We have clarified these issues by editing the figure and revising the legend as suggested in our new manuscript.6) Figure 3B: I suspect the markers on the gel are mistakenly labeled. For example, the experimental band of 380 bp is larger than the marker of 500 bp and the experimental band of 820 bp is smaller than the marker of 750 bp. Also, in Panel B, the gel of the 3' junctions: I don't understand how amplification from the donor shown in Panel A with T5F2 and T5R1 would give a band – is there some mistake here?loxP site, therefore a 730-bp product could be amplified in the lane with the donor as the template. We have edited the figure and revised the legend to clarify this issue.Sorry for the confusion due to our negligence in gel labeling. We have re-labeled the markers carefully, ensuring they are assigned at the correct position. Regarding the PCR result of the 3' junctions in Panel B, the sequences of both T5F2 and T5R1 primers indeed exist in the donor, flanking the upstream 7) In Figure 3C, I believe the images of dorsal view and ventral view have been reversed. I think the first column and bottom row picture is a ventral view. Please check – I may be wrong. I think the cross that produced the embryos in Panel C needs to be clearly explained and how to interpret the genotypes should be clearly explained. The images are difficult to interpret – what are we supposed to look at in the boxed insets? Explain in the legend please.Sorry for the confusion and thank you for the suggestion. We have carefully examined our original microscopic images and confirmed that the dorsal and ventral labeling are correct. We have indicated the expected genotypes in the figure and revised the legend in our new manuscript, as suggested.8) The images in Figure 3—figure supplement 1 are difficult to interpret. Again I suspect that if the authors told us the predicted genotypes and told us what we are supposed to be seeing in the outlined boxed areas, then it would be clearer.Sorry for the confusion and thank you for the suggestion. We have clarified these issues by editing the figure and revising the corresponding legend in our new manuscript, as suggested.Essential revisions:1) The revised manuscript is much improved, and additional data demonstrating feasibility of fully conditional knockout using the new cmlc2:CreERT2 line. However, one major issue remains. It is common practice to measure the expression of targeted loci in the "off" state in homozygotes. Data can be displayed in several different ways, exemplified by Figure 2 in Ni et al., 2012 reference and Figure 1D in the Grajevskaja et al., 2018. Figure 1A in Sugimoto et al., 2017 serves as a directly relevant example of from a recently published eLife paper describing a conditional knock-in allele. This is very important. As one can appreciate from error bars in Figure 2—figure supplement 1E, Figure 2—figure supplement 2L, Figure 3—figure supplement 1E and Figure 4—figure supplement 1G, it is impossible to distinguish between 90% and 99% mutagenesis efficiency in heterozygotes (55% vs. 51% of read-through transcript remaining in heterozygotes). In contrast, the difference between 10% and 1% levels of remaining wild-type transcript should be quite straightforward to reliably quantify in homozygotes. Results of such quantitative analysis may have an impact on the choice between your described polyA cassettes (tandem SV40/GBH vs. 8xSV40). Thus, qRT-PCR analyses must be performed on embryos homozygous for Cre-excised alleles.Ne/Netbx5a , Ne-1/Ne-1kctd10 , NeG/NeGtbx5a , and NeG/NeGsox10 , respectively.Thank you for the suggestions. We have measured the expression of all the four targeted loci in the “off” state from their corresponding homozygous mutant embryos by qRT-PCR. The results showed that the wild-type transcripts displayed at least a 12.5-fold reduction in the homozygous mutant embryos for each targeted allele. More precisely, their transcription level decreased to about 6.4%, 0.3%, 7.9% and 4.1% for 2) A second issue somewhat detracting from the overall quality of the work is occasionally difficult-to-read English. I would simply recommend that the final submission should be edited with the help of a professional editor.Thank you for the suggestions. Our current manuscript has been polished by the language editing service team of Nature Publishing Group, and we hope the new revision is now satisfactory.3) Sequences of primers used for qRT-PCR should be included, along with other primers, in Supplementary file 7.Sorry for the confusion. The sequences of primers used for PCR and qRT-PCR analyses were previously listed in the Supplementary file 7 and Supplementary file 8, respectively. Now we have combined all the primer sequence information into a single table as the new Supplementary file 7.4). Why is kctd10 deleted from the Abstract?kctd10 from the abstract due to word limitation. Now we have revised the Abstract and added kctd10 back to the new Abstract.Sorry for the confusion. We deleted 5) Subsection “High efficient generation of a dual-function KI allele at zebrafish tbx5a locus”: Need to show hEMX1 target site on the vector in Figure 1A, and corresponding sites in vector diagrams in subsequent figures.hEMX1 target site sequence on top of the donor vector, and depicted the site as a purple box in the diagram of the donor vector in our previous Figure 1A. Now we have improved the figure by adjusting the color of the box to better match the color of the above hEMX1 target site sequence, and labeling the box by adding the word “hEMX1 site” below it, as well as labeling the donor at its upper-left corner with “Donor vector”.Sorry for the confusion. We actually provided the hEMX1 site, as well as Figure 4—figure supplement 1D for the lamGolden site.Similarly, we have also revised all the other figures containing vector diagrams, including Figure 2—figure supplement 2D and Figure 3A for the


To facilitate assessment of the variability in sO2 and haemoglobin in tumours, for example in response to therapies, the baseline variability of these parameters was evaluated in subcutaneous head and neck tumours in mice, using a PAI system (MSOTinVision-256TF). Tumours of anaesthetized animals (midazolam-fentanyl-medetomidine) were imaged for 75 min, in varying positions, and repeatedly over 6 days. An increasing linear trend for average tumoural haemoglobin and blood sO2 was observed, when imaging over 75 min. There were no significant differences in these temporal trends, when repositioning tumours. A negative correlation was found between the percent decrease in blood sO2 over 6 days and tumour growth rate. This paper shows the potential of PAI to provide baseline data for assessing the significance of intra- and inter-tumoural variations that may eventually have value for predicting and/or monitoring cancer treatment response.Photoacoustic imaging (PAI) provides information on haemoglobin levels and blood oxygenation (sO Tempora2)  between days 1 and 6 of imaging, probably because they outgrew their blood supply before new vasculature could be formed. Slower growing tumours (n = 3) exhibited an increase or no change in sO2 [+12% and -1%], possibly because angiogenesis was occurring at a rate similar to the tumour growth rate. Interestingly, Tomaszewski et al. [2 (while O2 breathing) and ΔsO2 also decreased with tumour growth, from a 30 mm3 volume to time of culling. The CALR model used for this project has also been shown to develop rapidly and become hypoxic [In order to assess whether there was any dependence of tumour blood sOsurement  and the i et al.  used oxy hypoxic . This obA limitation of this study is that it evaluated the baseline variations in the photoacoustic signal for only one tumour type, a subcutaneous head and neck tumour model. Other tumour types might show different variation. For example, Tomaszewski et al.  showed t52 and HbT) and oxygen saturation over short (up to 75 min) and long (6 days) periods, and the variation with repositioning of the animal, in untreated subcutaneous head and neck tumours in nude mice. Time under anaesthesia is the largest source of temporal variability in the photoacoustic signal. To perform inter- and intra-animal comparison in longitudinal studies, for example when assessing the effects of treatment, ideally the imaging should be started at a fixed time point after anaesthesia and, if possible, sham-treated animals should be imaged at the same time points as the treated animals.This paper documents the variation in haemoglobin (Hb, HbOPositioning of the animal was found to influence image quality in a limited number of animals; when reconstruction artefacts or an unexpected absence in PAI signal are observed during the preview of the scan, the animal can be removed and re-positioned to obtain improved PAI signal. With a carefully planned experimental protocol, photoacoustic imaging could be used for assessing changes in tumour haemoglobin and oxygenation in longitudinal studies, including those designed to assess cancer therapies that may affect vasculature and, consequently, tumour oxygenation.All data and/or analysis performed in this study are available from the corresponding author on request.The authors declare that there are no conflict of interest.