Fluorescent in vivo editing reporter (FIVER): A novel multispectral reporter of in vivo genome editing
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Abstract
Advances in genome editing technologies have created opportunities to treat rare genetic diseases, which are often overlooked in terms of therapeutic development. Nonetheless, substantial challenges remain: namely, achieving therapeutically beneficial levels and kinds of editing in the right cell type(s). Here we describe the development of FIVER (fluorescent in vivo editing reporter) — a modular toolkit for in vivo detection of genome editing with distinct fluorescent read-outs for non-homologous end-joining (NHEJ), homology-directed repair (HDR) and homology-independent targeted integration (HITI). We demonstrate that fluorescent outcomes reliably report genetic changes following editing with diverse genome editors in primary cells, organoids and in vivo . We show the potential of FIVER for high-throughput unbiased screens, from small molecule modulators of genome editing outcomes in primary cells through to genome-wide in vivo CRISPR cancer screens. Importantly, we demonstrate its in vivo application in postnatal organ systems of interest for genetic therapies — retina and liver. FIVER will broadly help expedite the development of therapeutic genome surgery for many genetic disorders.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on September 21 2020, follows.
Summary
In this technical report, the authors use a previously-described mouse reporter construct to measure Cre-mediated recombination events, and repurpose it to examine Cas-induced double-strand break (DSB) repair gene editing events. All reviewers agree that the article will be of interest for the gene editing community and has the potential to make a significant addition to the field, in particular the delivery aspect in different experimental systems (cell culture, organoids, in vivo tissues). However, the reviewers also are in consensus that the manuscript is in need of additional experimentation to bolster the claims, in particular with regards to HITI and the complex events. Moreover, the authors …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on September 21 2020, follows.
Summary
In this technical report, the authors use a previously-described mouse reporter construct to measure Cre-mediated recombination events, and repurpose it to examine Cas-induced double-strand break (DSB) repair gene editing events. All reviewers agree that the article will be of interest for the gene editing community and has the potential to make a significant addition to the field, in particular the delivery aspect in different experimental systems (cell culture, organoids, in vivo tissues). However, the reviewers also are in consensus that the manuscript is in need of additional experimentation to bolster the claims, in particular with regards to HITI and the complex events. Moreover, the authors need to address additional points as detailed below, many of which require only clarification, elaboration and text changes but not additional data. The claim in Figure 4 is not well corroborated and may be dropped unless a pilot screen and its results are presented in the revision. The claim in Figure 8 is not essential and may be dropped for better elaboration in an independent study. Below is a summary of the comments listing the essential revisions required for a revised manuscript.
Essential Points
The title should be changed. First, the use of in vivo may imply to some that the system is only for animals, which would short-sell its value. Second, given that the FIVER system is essentially the creative but simple use of dual CRISPR/Cas targeting on top of an already existing system (mT/mG Cre reporter mouse), the use of a novel acronym (FIVER) is not merited.
The Abstract should include the fact that this represents a previously-described Cre/Lox reporter repurposed for gene editing analysis. This information is in the text, but more transparency is required. The repurposing of a previously-described LoxP reporter assay for gene editing does not constitute a novel reporter assay. Moreover, the abstract should highlight the gene delivery advance.
The text requires elaboration and comparisons to other delivery approaches in the literature for each tissue examined. For example, it is unclear whether the efficiency of the delivery of Cas/sgRNAs to the retina in Figure 7H is expected based on other studies of gene delivery to this tissue. Namely, is it more difficult to achieve editing in this tissue, compared to introduction of a fluorescent transgene? If this has not been done, it does not need to be done here, but such comparisons with experiments in the literature will reinforce the utility of the approach.
The HDR reporter presentation could be clarified and the assay has some limitations that should be discussed. Figure 1a is difficult to follow, because the repair templates are not shown. It is suggested to show at least the minicircle template and the targeted integration. In general, please strive that figures are understandable without consulting the text.
Overall the frequencies of HDR are very low, but this is expected due to the design of the assay. Since two tandem DSBs are induced, NHEJ using the distal DSB ends causes loss of both cut sites, and hence is likely highly favored over terminal repair event. In contrast, most gene editing events involving HDR are induced by a single DSB, for which NHEJ recreates the cut site, and hence is a futile repair event. Namely, HDR is also promoted by the persistent nature of single Cas9-induced DSBs, which are inhibited in this assay by the second DSB. Also, the HDR event here requires a relatively long gene conversion tract, which is also not necessarily the goal of therapeutic gene editing. Accordingly, it is unclear whether this assay will be particularly useful for studying HDR.
It is inaccurate to state this is the first in vivo gene editing reporter for HDR. The DR-GFP mouse was established and used to examine HDR frequencies in mouse tissues over 7 years ago (PMID: 23509290). The text should be changed and this information should be included in the manuscript.
The validation of HITI editing and proposed applications are not at the level of Figures 1-3 validating endjoining or HDR events. NGS or equivalent quantitative techniques should be applied also here. This is especially true for the embryo editing application (Fig. 7) and the independent locus editing correlation (Fig. 8). For example, the very low levels of mosaicity during embryo editing is particularly surprising, since other papers have indicated that this is a major problem by sequence-based methods and phenotypic outcomes (e.g. tyrosinase editing for coat color). Have the authors validated the frequency of BFP+ events that are bona fide integration events at the target locus vs. random integration? This could be done, for example, by showing that the DSB at the target locus is required for BFP+ cells. BFP-minicircle transfections are used as negative controls, but do these also lack the DSB in the BFP-minicircle? Namely, the appropriate negative control should be BFP-minicircle + the DSB cutting the minicircle, but without the DSB cutting the target locus. This requires extensive new validation experiments that are essential for a revision.
To improve the utility of the assay, the "unexpected outcome" of editing at the reporter locus (i.e. +tdTomato/+EGFP) must be investigated further with new experimentation to fully understand the structure of the event and potentially deduce the mechanism(s) leading to it.
Figure 4 and associated main text. The claim that the reporter assay can be used in drug screens is not well supported. Either a proof-of-concept screen should be conducted or otherwise this claim should be removed.
The small molecule SCR7 that has been reported to target DNA Ligase 4 (Lig4) is discredited (PMID: 27235626) and this should be discussed and indicated in any figure, if this section were to remain in the manuscript.
- The claim made in Figure 8 that editing at the reporter locus corresponds to editing at another independent locus requires further evidence. Controls for cutting efficiency are lacking and more loci need to be tested. In the context of the dual-editing (HITI at mT/mG and Zmynd10), it should furthermore be evaluated whether integration of the n.TagBFP occurs at the Zmynd10 locus and vice versa. The comments must be addressed experimentally, if this claim were to remain in the revised manuscript.
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