Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility

Reviewer #1 (Public review):

Summary:

In this work, a screening platform is presented for rapid and cost-effective screening of candidate genes involved in Fragile Bone Disorders. The authors validate the approach of using crispants, generating FO mosaic mutants, to evaluate the function of specific target genes in this particular condition. The design of the guide RNAs is convincingly described, while the effectiveness of the method is evaluated to 60% to 92% of the respective target genes being presumably inactivated. Thus, injected F0 larvae can be directly used to investigate the consequences of this inactivation.

Skeletal formation is then evaluated at 7dpf and 14dpf, first using a transgenic reporter line revealing fluorescent osteoblasts, and second using alizarin-red staining of mineralized structures. In general, it appears that the osteoblast-positive areas are more often affected in the crispants compared to the mineralized areas, an observation that appears to correlate with the observed reduced expression of bglap, a marker for mature osteoblasts, and the increased expression of col1a1a in more immature osteoblasts.

Finally, the injected fish (except two lines that revealed high mortality) are also analyzed at 90dpf, using alizarin red staining and micro-CT analysis, revealing an increased incidence of skeletal deformities in the vertebral arches, fractures, as well as vertebral fusions and compressions for all crispants except those for daam2. Finally, the Tissue Mineral Density (TMD) as determined by micro-CT is proposed as an important marker for investigating genes involved in osteoporosis.

Taken together, this manuscript is well presented, the data are clear and well analyzed, and the methods are well described. It makes a compelling case for using the crispant technology to screen the function of candidate genes in a specific condition, as shown here for bone disorders.

Strengths:

Strengths are the clever combination of existing technologies from different fields to build a screening platform. All the required methods are comprehensively described.

Weaknesses:

One may have wished to bring one or two of the crispants to the stage of bona fide mutants, to confirm the results of the screening, however, this is done for some of the tested genes as laid out in the discussion.

Reviewer #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather than question/hypothesis-driven.

(2) Some of the measurements are inadequately normalized and not as specific to bone as suggested:

(a) The measurements of surface area covered by osteoblasts or mineralized bone (Figure 1) should be normalized to body size. The authors note that such measures provide "insight into the formation of new skeletal tissue during early development" and reflect "the quantity of osteoblasts within a given structure and [is] a measure of the formation of bone matrix." I agree in principle, but these measures are also secondarily impacted by the overall growth and health of the larva. The surface area data are normalized to the control but not to the size/length of each fish - the esr1 line in particular appears quite developmentally advanced in some of the images shown, which could easily explain the larger bone areas. The fact that the images in Figure S5 were not all taken at the same magnification further complicates this interpretation.

(b) Some of the genes evaluated by RT-PCR in Figure 2 are expressed in other tissues in addition to bone (as are the candidate genes themselves); because whole-body samples were used for these assays, there is a nonzero possibility that observed changes may be rooted in other, non-skeletal cell types.

(3) Though the assays evaluate bone development and quality at several levels, it is still difficult to synthesize all the results for a given gene into a coherent model of its requirement.

(4) Several additional caveats to crispant analyses are worth noting:

(a) False negatives, i.e. individual fish may not carry many (or any!) mutant alleles. The crispant individuals used for most assays here were not directly genotyped, and no control appears to have been used to confirm successful injection. The authors therefore cannot rule out that some individuals were not, in fact, mutagenized at the loci of interest, potentially due to human error. While this doesn't invalidate the results, it is worth acknowledging the limitation.

(b) Many/most loci identified through GWAS are non-coding and not easily associated with a nearby gene. The authors should discuss whether their coding gene-focused pipeline could be applied in such cases and how that might work.

Reviewer #3 (Public review):

Summary:

The manuscript "Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility" describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). The authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observed varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes.

A major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested.

A major weakness is a lack of new insights into underlying mechanisms that may contribute to disease phenotypes, nor any clear commonalities across gene sets. This was most evident in the qRT-PCR analysis of select skeletal developmental genes, which all showed varied changes in fold and direction, but with little insight into the implications of the results.

Ultimately, the authors were able to show their approach is capable of connecting candidate genes with perturbation of skeletal phenotypes. It was surprising that all four GWAS candidate genes (which presumably were lower confidence) also produced a result. These authors have previously demonstrated that crispants recapitulate skeletal phenotypes of stable mutant lines for a single gene, somewhat reducing the novelty of the study.