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  1. Author Response:

    Reviewer #2 (Public Review):

    There is now a considerable body of knowledge about the genetic and cellular mechanisms driving the growth, morphogenesis and differentiation of organs in experimental organisms such as mouse and zebrafish. However, much less is known about the corresponding processes in developing human organ systems. One powerful strategy to achieve this important goal is to use organoids derived from self-renewing, bona fide progenitor cells present in the fetal organ. The Rawlins' lab has pioneered the long-term culture of organoids derived from multipotent epithelial progenitors located in the distal tips of the early human lung. They have shown that clonal cell "lines" can be derived from the organoids and that they capable of not only long-term self-renewal but also limited differentiation in vitro or after grafting under the kidney capsule of mice. Here, they now report a strategy to efficiently test the function of genes in the embryonic human lung, regardless of whether the genes are actively transcribed in the progenitor cells. The strengths of the paper are that the authors describe a number of different protocols (work-flows), based on Crisper/Cas9 and homology directed repair, for making fluorescent reporter alleles (suitable for cell selection) and for inducible over-expression or knockout of specific genes. The so-called "Easytag" protocols and results are carefully described, with controls. The work will be of significant interest to scientists using organoids as models of many human organ systems, not just the lung. The weaknesses are that they authors do not show that their lines can undergo differentiation after genetic manipulation, and therefore do not provide proof of principle that they can determine the function in human lung development of genes known to control mouse lung epithelial differentiation. It would also be of general interest to know whether their methods based on homologous recombination are more accurate (fewer incorrect targeting events or off target effects) than methods recently described for organoid gene targeting using non homologous repair.

    We thank Reviewer #2 for capturing the key advances of our toolbox for understanding gene function using a tissue organoid system and the constructive suggestions for the manuscript.

    We agree with the Reviewer that it would strengthen the current manuscript if we could differentiate the genetically targeted organoids. Therefore, as a proof of concept, we have successfully differentiated the SOX9 reporter organoids into the alveolar lineage (New figure: Figure 2-figure supplement 1g, shown above). We have also tested the dual SMAD inhibition approach recently reported for basal cell differentiation (Miller et al., 2020). However, this has led to massive cell death even in WT organoids (data not shown). We reason that this might be because our organoids are ~8 pcw, whereas in the literature ~12 pcw organoids were used. We believe that efficient airway differentiation will take a long time to optimise for our organoids and is therefore beyond the scope of this manuscript.

    In regard to the Easytag workflow in comparison with the recent CRISPR-HOT method using non-homologous end joining (Artegiani et al., 2020), we consider our approach as a complement to the CRISPR-HOT approach. This can be reflected in the following points: (1) The Organoid Easytag workflow allows precise N-terminal tagging of endogenous genes, exemplified by N-terminal tagging of ACTB. This is not possible using CRISPR-HOT as large pieces of plasmid DNA would disrupt the targeted gene; (2) The Organoid Easytag workflow is based on HDR and the efficient insertion sites for exogenous genes are within a ~30-bp window of the gRNA cleavage sites (Kwart et al., 2017), which gives more flexibility for choosing gRNAs compared with CRISPR-HOT tagging; (3) The Organoid Easytag workflow gives researchers more control of where and how the targeted sites can be modified, and offers a minimal change to the targeted genomic region, whereas CRISPR-HOT introduces large pieces of backbone plasmids, which potentially increases the risk of gene dysregulation. However, HDR requires cells to be at the G2/M phase of the cell cycle, therefore heavily relying on fast cycling cells to gain the most efficient targeting. CRISPR-HOT has the great advantage of not depending on a specific cell cycle stage and therefore being more efficient in slow cycling cells. With this said, we do believe that the efficiency would very much rely on the context, including the cell type used and locus targeted, as a recent report suggested targeting efficiency is influenced also by genomic context (Schep et al., 2021).

    In summary, when N-terminal tagging, minimal changes and precise control of targeting is desired, Organoid Easytag is more favourable; whereas when targeting slowly cycling cells, CRISPR-HOT has its strength. Therefore, we consider these two methods as complementary approaches that will both be of benefit to organoid-based research. We have summarised this comparison into a simple table (New table: Figure 2-figure supplement 5f)

    Figure 2-figure supplement 5(f). A comparison of Organoid Easytag and CRISPR-HOT methods (Artegiani et al., 2020).

    Reviewer #3 (Public Review):

    Sun et al have assembled, modified, and applied a series of existing gene editing tools to tissue-derived human fetal lung organoids in a workflow they have termed "Organoid Easytag". Using approaches that have previously been applied in iPSCs and other cell models in some cases including organoids, the authors demonstrate: 1) endogenous loci can be targeted with fluorochromes to generate reporter lines; 2) the same approach can be applied to genes not expressed at baseline in combination with an excisable, constitutively active promoter to simplify identification of targeted clones; 3) that a gene of interest could be knocked-out by replacing the coding sequence with a fluorescent reporter; 4) that knockdown or overexpression can be achieved via inducible CRISPR interference (CRISPRi) or activation (CRISPRa). In the case of CRISPRi, the authors alter existing technology to lessen unwanted leaky expression of dCas9-KRAB. While these tools have previously been applied in other models, their assembly and demonstrated application to tissue-derived organoids here could facilitate their use in tissue-derived organoids by other groups.

    Limitations of the study include:

    1. is demonstrated application of these technologies to a limited set of gene targets;
    1. a lack of detail demonstrating the efficiency and/or kinetics of the approaches demonstrated.

    While access to human fetal lung organoids is likely not available to many or most researchers, it is probable that the principles applied here could carry over to other organoid models.

    We thank the Reviewer for accurately summarising the details of our manuscript and positive comments on its potential to facilitate tissue-derived organoid related research. We are very grateful for the Reviewer’s detailed and constructive comments to help strengthen our manuscript.

    In regard to the limitations pointed out by Reviewer #3, we have systematically tested the kinetics of the inducible CRISPRi knockdown effect and its reversibility using CD71 and SOX2 (New figure: Figure 3-figure supplement 2). At the same time, we have generated SOX9 reporter human foetal intestinal organoids using the Easytag workflow to further demonstrate it can be applied to another organoid system. As suggested by Reviewer #3, we also attempted to implement the inducible CRISPRi system in HBECs. However, due to their sensitivity to lentiviral transduction, infected HBECs died shortly after transduction with gRNA lentivirus. We believe that further optimisation of DNA delivery approach is required for implementation of the inducible CRISPRi/CRISPRa systems in HBECs (perhaps nucleofection and PiggyBac-based vectors).

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  2. Reviewer #3 (Public Review):

    Sun et al have assembled, modified, and applied a series of existing gene editing tools to tissue-derived human fetal lung organoids in a workflow they have termed "Organoid Easytag". Using approaches that have previously been applied in iPSCs and other cell models in some cases including organoids, the authors demonstrate: 1) endogenous loci can be targeted with fluorochromes to generate reporter lines; 2) the same approach can be applied to genes not expressed at baseline in combination with an excisable, constitutively active promoter to simplify identification of targeted clones; 3) that a gene of interest could be knocked-out by replacing the coding sequence with a fluorescent reporter; 4) that knockdown or overexpression can be achieved via inducible CRISPR interference (CRISPRi) or activation (CRISPRa). In the case of CRISPRi, the authors alter existing technology to lessen unwanted leaky expression of dCas9-KRAB. While these tools have previously been applied in other models, their assembly and demonstrated application to tissue-derived organoids here could facilitate their use in tissue-derived organoids by other groups.

    Limitations of the study include:

    1. is demonstrated application of these technologies to a limited set of gene targets;

    2. a lack of detail demonstrating the efficiency and/or kinetics of the approaches demonstrated.

    While access to human fetal lung organoids is likely not available to many or most researchers, it is probable that the principles applied here could carry over to other organoid models.

    Was this evaluation helpful?
  3. Reviewer #2 (Public Review):

    There is now a considerable body of knowledge about the genetic and cellular mechanisms driving the growth, morphogenesis and differentiation of organs in experimental organisms such as mouse and zebrafish. However, much less is known about the corresponding processes in developing human organ systems. One powerful strategy to achieve this important goal is to use organoids derived from self-renewing, bona fide progenitor cells present in the fetal organ. The Rawlins' lab has pioneered the long-term culture of organoids derived from multipotent epithelial progenitors located in the distal tips of the early human lung. They have shown that clonal cell "lines" can be derived from the organoids and that they capable of not only long-term self-renewal but also limited differentiation in vitro or after grafting under the kidney capsule of mice. Here, they now report a strategy to efficiently test the function of genes in the embryonic human lung, regardless of whether the genes are actively transcribed in the progenitor cells. The strengths of the paper are that the authors describe a number of different protocols (work-flows), based on Crisper/Cas9 and homology directed repair, for making fluorescent reporter alleles (suitable for cell selection) and for inducible over-expression or knockout of specific genes. The so-called "Easytag" protocols and results are carefully described, with controls. The work will be of significant interest to scientists using organoids as models of many human organ systems, not just the lung. The weaknesses are that they authors do not show that their lines can undergo differentiation after genetic manipulation, and therefore do not provide proof of principle that they can determine the function in human lung development of genes known to control mouse lung epithelial differentiation. It would also be of general interest to know whether their methods based on homologous recombination are more accurate (fewer incorrect targeting events or off target effects) than methods recently described for organoid gene targeting using non homologous repair.

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  4. Reviewer #1 (Public Review):

    The authors demonstrate applications including fluorescent marking of membranes with GFP or monomeric RFP, reporter alleles for convenient assessment of differentiation status based on fluorescence, and targeted gene knockout. They also demonstrate conditional gene knockdown and induction with tight control achieved by engineering a protein destabilizing domain. The design of the constructs is clever and imparts the ability to leverage iterative FACS to enrich successfully targeted cells, particularly useful when targeting alleles that are not actively expressed by the progenitors. The work is well done and clearly presented.

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  5. Evaluation Summary:

    In this paper Sun and colleagues aimed to demonstrate the feasibility of using CRISPR-based gene editing techniques applied to tissue-derived human fetal lung organoids. While previous studies have used CRISPR-Cas9 to perform knock-in or knock-out studies in organoids (such as intestinal, hepatic or tumor organoids), this is the first report to apply these tools to a tissue-derived lung organoid model. A major strength of this report is the additional use of CRISPRi and CRISPRa technologies. The work is well done, clearly presented and makes an important contribution to the literature.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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