Mutations in the human CYP21A2 gene are the most frequent cause of congenital adrenal hyperplasia (CAH), characterized by 21-hydroxylase deficiency and consequent impaired synthesis of cortisol and aldosterone. In mice, the homologous Cyp21a1 gene encodes a functionally analogous enzyme, making it a suitable target for generating a knockout model that replicates the molecular mechanisms of the disease. This study describes the design and experimental validation of two single-guide RNA (sgRNA) pairs targeting exons 1–10 of the Cyp21a1 gene using the CRISPR/Cas9 system. Guide RNA efficacy was assessed in vitro in murine B16 cells and in vivo following microinjection of ribonucleoprotein (RNP) complexes into zygotes and subsequent analysis of blastocyst-stage embryos. The selected guides enable the deletion of a major portion of the coding sequence of the gene, as confirmed by PCR analysis and sequencing. These results demonstrate the feasibility of using the chosen sgRNAs to establish a Cyp21a1–/– knockout mouse line, which will serve as a promising model for preclinical studies of gene therapy for CAH.
Antonova et al. (Fri,) studied this question.