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Abstract <p>Mutations in the human <i>CYP21A2</i> 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 <i>Cyp21a1</i> 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 <i>Cyp21a1</i> 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 <i>Cyp21a1</i>–/– knockout mouse line, which will serve as a promising model for preclinical studies of gene therapy for CAH.</p>

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

  • 期刊论文

  • 发表日期：

    2026年5月5日

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  • E. N. Antonova,
  • E. E. Uldanova,
  • A. S. Trifonova,
  • E. D. Piven,
  • O. V. Glazova,
  • N. Sakr,
  • N. V. Rudev,
  • M. V. Vorontsova,
  • O. N. Mityaeva,
  • P. Y. Volchkov

摘要

Abstract

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.