A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish

  1. Yueyang Wang
  2. Alan Y. Hsu
  3. Eric M. Walton
  4. Sung Jun Park
  5. Ramizah Syahirah
  6. Tianqi Wang
  7. Wenqing Zhou
  8. Chang Ding
  9. Abby Pei Lemke
  10. GuangJun Zhang
  11. David M. Tobin
  12. Qing Deng

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Abstract

CRISPR/Cas9-based tissue-specific knockout techniques are essential for probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of single guide (sg)RNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 genes restores neutrophil motility in the corresponding knockout background. Moreover, active Rac and force-bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.

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  1. Version published to 10.1242/jcs.258574
  2. eLife

    ###Reviewer #3:

    Wang et al describe a tissue specific knockout system to target neutrophil specific genes. Tissue specific knockout system is an important tool to study gene function in specific tissues. To the best of my knowledge there are only four major publications describing the tissue specific knockouts in zebrafish, two of them are not acknowledged by the authors.

    In this manuscript, authors used neutrophil specific promoter to drive the expression of Cas9, and ubiquitous promoter for sgRNA expression or vice versa. The authors have previously published a similar paper describing a transgenic construct (Tg(lyzC:nls-cas9-2A-mCherry/U6a:polg sgRNA)) expressing Cas9 as well as sgRNAs from the single construct. Authors claimed that the knockout efficiency drops significantly when the knockout line is crossed with other lines that use the neutrophil-specific promoter possibly due to the presence of another construct driven by the same neutrophil-specific promoter in the genome competes with the transcriptional factors for Cas9 expression and reduces Cas9 protein to a level that is not sufficient for efficient knockout.

    In this manuscript authors created a sgRNA-resistant rescue construct, and incorporate biosensors into the knockout line for live imaging in the context of the cell-specific knockout, and studied the function of Rac2 and Cdk2.

    This manuscript does not offer any further advances other than showing the tissue specific rescue, and subcellular localization of Rac activation in wild-type and rac2-knockout neutrophils.

    There is no evidence that this strategy is better than the previously published method, the quantification of knockout efficiency is absent.

  3. eLife

    ###Reviewer #2:

    In the manuscript "A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish" by Wang et al, the authors describe methods for targeted inactivation of genes in a cell-type specific fashion, in this case in neutrophils in zebrafish embryos, and use this tool to examine the role of rac2 in neutrophil motility. The overall goal of broadening the ability to target tissue-specific gene inactivation is laudable and an ongoing need in the zebrafish toolbox, as is the goal of developing an increased understanding of motility regulation in neutrophils, as evaluated here in a series of quite stunning motion-tracking videos. Unfortunately, the current manuscript does not appear to advance the technology, nor evaluate it in sufficient depth, nor reveal sufficient new biology in regards to neutrophils/rac2.

    Major Points:

    1. With the title "A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish", the manuscript seems to be targeting a "technology" aim. As the authors cite, they have already published a neutrophil-specific CRIPSR/Cas9-based knockout tool in their DMM, 2018 manuscript. The addition of the crystallin reporter in the current manuscript is a convenient method for tracking the cas9 portion of the transgene, but this is a modest alteration to the existing technology.

    2. While billed as a neutrophil-specific gene-disruption technology, the authors do not show genome sequence of a mutated/disrupted rac2 gene. They have previously done this in the DMM 2018 paper, so should be feasible. Disruption of neutrophil motility is being used as a proxy read-out for rac2 disruption, but it seems that, as currently billed, the study should show neutrophil-specific disruption of rac2. The neutrophil-specific rescue experiments are very nice, but fail to show that the targeted gene disruption is limited to neutrophils, only that the gene disruption includes neutrophils. This could be of concern in a stable transgene context as well since transgenes can exhibit ectopic gene expression (i.e. not limited to neutrophils), and this cannot be tracked with the un-tagged CAS9 in the construct.

    3. At the outset, it is expected that disruption of rac2 would lead to neutrophil motility disruption and changes in F-actin dynamics using this tool as previously described in Deng et al, "Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue", Dev Cell, 2011. As a proof of concept for the ability of targeting a gene in neutrophils, this makes sense to evaluate a well-studied pathway, but it is not clear if this expands on the understanding of rac2/control of actin dynamics and neutrophil motility, or if the newly described targeting vectors allow for an analysis that was not previously possible.

    4. The ribozyme approach described in Figure 6 seems perhaps most novel as an approach to target tissue-specific inactivation of a gene, but to truly nail down the technology, this would seem to require again some analysis of (a) the specific genomic lesions induced by the combination of ubiquitous CAS9 and tissue-specific gRNA and (b) some assessment of the specificity to neutrophils (i.e. are these mutations generated in other cell types?).

  4. eLife

    ###Reviewer #1:

    Summary:

    Wang et al. utilize in their manuscript two trangenic lines to tissue-specifically knockout the rac2 gene in neutrophils. While technically CRIPSR-Cas9 has been well established, tissue-specific knockouts in zebrafish are missing in the field. Therefore, the manuscript of Wang et al. is highly timely and would help advance the field further; however, the manuscript and figures would greatly benefit from thorough editing and rewriting as outlined below.

    Major comments:

    Wang et al. base all their conclusions on observations of the targeted cells, and do not show any sequenced alleles of the neutrophil cells to verify that indels occurred. To go forward with the results, including sequences of the targeted alleles is crucial. Therefore, the manuscript would greatly benefit from including these basic allele confirmations, before drawing scientific conclusions about the efficacy of the system.

    1. Line 100 onwards. "To test the efficiency of the gene knockout using this system, we injected the F2 embryos of the Tg(lyzC:cas9, cry:GFP) pu26 101 line with the plasmids carrying rac2 sgRNAs or ctrl sgRNAs 102 for transient gene inactivation. The sequences of the sgRNAs are described in Fig. 1C, D. A 103 longer sequence with no predicted binding sites in the zebrafish genome was used as a control 104 sgRNA (Fig. 1D). As expected, we observed significantly decreased neutrophil motility in larvae of Tg(lyzC:cas9, cry:GFP) pu26 105 fish transiently expressing sgRNAs targeting rac2 (Fig. 106 1E, F and Movie S1), indicating that sufficient disruption of the rac2 gene had been achieved."

    Please include sequenced alleles from rac2 in neutrophil cells. "Significantly decreased neutrophil motility" is not an indicator that rac2 in neutrophil cells is mutated. Only sequenced alleles are.

    1. Line 107 onwards. "To test the knockout efficiency in stable lines, we generated transgenic lines of Tg(U6a/c: ctrl sgRNAs, lyzC:GFP) pu27 or Tg(U6a/c: rac2 sgRNAs, lyzC:GFP) pu28 108 , crossed the F1 fish with Tg(lyzC:cas9, cry:GFP) pu26 109 and quantified the velocity of neutrophils in the head mesenchyme 110 of embryos at 3 dpf. A significant decrease of motility was observed in the neutrophils 111 expressing Cas9 protein and rac2 sgRNAs (Fig. 1G, H and Movie S2)."

    Also here, "a significant decrease of motility" doesn't mean the rac2 gene in neutrophils is mutated. See point 1.

    Summarizing, the authors are advised to include this basic, but necessary and very important information in their manuscript instead of drawing conclusions from their observations. Otherwise, it stays unclear if everything Wang et al. observe is really due to indels in the rac2 gene, and not some other side effect of the system.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    As you can see from the reviewer comments attached below, all reviewers appreciated the approach you took for neutrophil-specific gene disruption, as such tissue-specific tools remain greatly missing in the field. Nonetheless, the reviewers all agreed that your phenotype description is insufficient to warrant the claims of the study. In particular, the lack of sequence verification of the claimed Cas9-induced mutagenesis has been picked up by all reviewers. We hope the reviewer comments are instrumental for refining your work.

  6. Version published to 10.1101/2020.07.27.223008v1 on bioRxiv