Background: Why build variant-specific disease models

A study on establishing an in vitro model of FGFR2-related craniosynostosis found that prime editing efficiency varied with cell type, target site, and cell attachment status. Human MSC-TERT cells, which have osteogenic potential, were more resistant to editing than the comparator HEK293FT cells. The study focused on conditions for building cell models to investigate disease-associated variants, not on therapeutic effects in patients.

Craniosynostosis is a rare congenital skeletal disorder in which the sutures between skull bones close prematurely. It is associated with variants in more than 60 genes. Variant-specific in vitro models allow researchers to study these variants without relying on patient-derived material or the expression of externally introduced genes. Building such models requires determining how efficiently editing occurs in the cells of interest.

The researchers applied prime editing, a modified form of the CRISPR-Cas9 gene-editing system, to MSC-TERT, an immortalized human bone marrow-derived mesenchymal stem cell line. The approach aimed to establish a basis for building models of different FGFR2 variants in cells with the potential to form bone.

Key findings: Conditions that improved editing in comparator cells did not work the same way

Prime editing was more difficult in MSC-TERT than in HEK293FT. The two cell types also responded differently when the researchers added dnMLH1 and engineered prime editing guide RNA (epegRNA). Editing efficiency increased in HEK293FT, but the same improvement was not observed in MSC-TERT. These findings indicate that conditions effective in comparator cells cannot be assumed to work in cells with osteogenic potential.

Editing efficiency also varied by genomic target site. Higher efficiency was reported in non-adherent cells than in cells attached to the culture surface. However, the provided abstract does not describe a mechanism for this difference or give quantitative editing rates for each cell type.

In non-adherent HEK293FT cells that were isolated and cultured separately, editing continued for four days. This observation is limited to those cells and culture conditions. There is no basis for interpreting it as evidence that editing continued for the same period in MSC-TERT or that a disease model with long-term stability was established.

Implications and outlook: Establishing conditions suited to each cell type remains a challenge

This study provides a basis for using prime editing to build variant-specific in vitro models of FGFR2 and applying the approach to MSC-TERT. In particular, differences across cell types in editing resistance and in the effects of conditions intended to improve editing are constraints to consider in model development. Researchers need both to select cells suited to their aims and to validate editing conditions in those cells.

The available material consists of the journal article’s bibliographic information and abstract. The specific target variants, editing rates, sample size, and off-target measurements could not be confirmed. The evidence therefore does not support conclusions that a specific variant was fully corrected to the normal sequence or that osteogenic function was normalized. This also cannot be regarded as a study evaluating therapeutic effects in patients. The achievement supported here is the evaluation of editing conditions for building in vitro disease models.

Source: The CRISPR Journal article abstract and bibliographic information, article DOI.