Design and Efficacy Evaluation of CRISPR/Cas9 Guide RNAs for Knockout of the Cyp21a1 Gene in Mouse Cells and Embryos

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.