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

OBJECTIVES: Osteogenesis imperfecta (OI), characterized by skeletal
deformities, is a genetically heterogeneous connective tissue disorder
mainly caused by autosomal dominant mutations in collagen type 1.
Mutations in non-collagen genes are mostly associated with recessive
forms of OI and encode proteins involved in osteoblast differentiation, bone mineralization and collagen processing. However, the genetic
spectrum of OI is expanding and the underlying pathogenic mechanisms
are often not yet known. To quickly gain insight into these mechanisms,
we aimed to develop a rapid and cost-effective approach for in vivo
functional validation of OI-causing genes, using the zebrafish model
system. This model is attractive to study skeletal diseases due to its easy
genetic manipulation and genetic similarity to humans.
METHODS: We used the so-called crispant screening approach, based on
CRISPR/Cas9 technology, in order to phenotype directly in FO mosaic
founder zebrafish. Crispants significantly reduce model generation time,
enabling the validation of a large set of genes in a short period of time.
RESULTS: We selected a panel of 6 OI genes (creb3l1, ifitm5, mbtps2,
sec24d, serpinf1 and sparc) and micro-injected CRISPR/Cas9
components (gRNA/Cas9) targeting the gene of interest, in one-cell
stage zebrafish embryos with an osteoblast-specific Tg(osx:Kaede)
transgenic background. NGS analysis revealed out-of-frame efficiencies
higher than 70%, indicating a high fraction of knock-out alleles and thus
resembling a stable knock-out model. Phenotypic analysis was
performed at 14 and 90 days after fertilization, though fluorescence
microscopy (osteoblasts), alizarin red bone staining and micro-CT
analysis for quantitative analysis of the skeleton. Crispants for creb3l1,
coding for OASIS, which has an important role in osteoblast
differentiation during bone development, show skeletal abnormalities,
such as vertebral fusions, callus formation in the vertebral arches
indicating previous fractures and ectopic mineralization at 90 dpf. These
investigations are also ongoing for the remaining 5 genes.
CONCLUSION: Taken together, we showed that crispant screening in
zebrafish is a promising approach for rapid functional screening of OI
candidate genes. Moreover, the crispants have the potential to provide
new insights into the role of these genes in skeletal biology and can be
used as a tool for osteogenic compound screening.