Correction of genetic dwarfism (achondroplasia) using CRISPR base-editing — ASN Events
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Correction of genetic dwarfism (achondroplasia) using CRISPR base-editing (#116)

Julian Chu 1 2 3 , Alexandra O'Donohue 1 2 4 , Samantha Ginn 3 5 , Aaron Schindeler 1 2 4
  1. Bioengineering and Molecular Medicine Laboratory, Westmead Institute of Medical Research and The Children’s Hospital at Westmead, Westmead, NSW, Australia
  2. Centre of Diabetes, Obesity and Endocrinology, Westmead Institute of Medical Research, Westmead, NSW, Australia
  3. School of Medical Science, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia
  4. School of Chemical and Biomolecular Engineering, Faculty of Engineering, The University of Sydney, Camperdown, NSW, Australia
  5. Gene Therapy Unit, the Children's Medical Research Institute, Westmead, NSW, Australia

Achondroplasia (ACH) is the most common form of genetic dwarfism and is inherited in an autosomal dominant manner. Approximately 97% of cases are caused by a single‑nucleotide mutation (c.1138G>A) in the fibroblast growth factor receptor 3 (FGFR3) gene, resulting in constitutive receptor activation and impaired endochondral bone growth. CRISPR base editing (BE) is an emerging genome‑editing approach that enables precise correction of pathogenic point mutations without inducing double‑stranded DNA breaks. By utilising an adenine base editor (ABE) capable of A→G conversion, the causative FGFR3 mutation underlying ACH can be precisely corrected.

HEK293T cells were first engineered to harbour the FGFR3 c.1138G>A point mutation using CRISPR‑Cas9 nickase (Cas9n). Initial correction of the mutant locus was performed using the nSpRY‑ABE8e base editor, followed by iterative optimisation using alternative SpRY‑ABE8e variants. Split‑intein versions of the ABE constructs were rationally designed and mutant locus correction was evaluated using a dual vector approach. Editing efficiency, bystander editing, and base conversion at the top-ten off‑target sites were quantified using next‑generation sequencing.

Generation of the FGFR3 c.1138G>A knock‑in achieved 37% efficiency, enabling isolation of a homozygous mutant clone. nSpRY‑ABE8e yielded 70.5 %on‑target editing but 67.0% bystander and WT allele editing (Figure 1). Iterative optimisation with SpRY‑ABE8e variants improved precision, with SpRY‑HF1‑ABE8eF148A achieving 53.5% on‑target editing and only 7.5% bystander edits. Notably, split‑intein constructs preserved editing efficiency while eliminating detectable bystander editing.

Collectively, these findings demonstrate the feasibility of precise CRISPR-ABE–mediated correction of the causative FGFR3 mutation in ACH. Ongoing work will focus on evaluating adeno‑associated viral vectors (AAV) as a delivery platform by assessing candidate serotypes in growth plate chondrocytes. Therapeutic efficacy will be subsequently tested in an ACH murine model featuring a humanized FGFR3 c.1138G>A allele. These studies will advance AAV-ABE vector technology toward a clinical gene therapy treatment for dwarfism.

69eb0b6f57d2c-Combined.png

Figure 1. Rationally optimised CRISPR-ABEs were used to optimise editing efficiency in ACH mutant cells. nSpRY-ABE8e yielded high on-target editing but unwanted bystander and WT editing. The split-intein version of SpRY-HF1-ABE8eF148A remained efficient and ablated bystander and WT editing.