Standardizing CRISPR-Cas13 knockdown technique to investigate the role of cdh2 gene in pituitary development through growth hormone expression and transcription factors.

INTRODUCTION

Congenital hypopituitarism (CH) is characterized by the deficiency of pituitary hormones. Among CH patients, 85% lack a molecular diagnosis. Whole Exome Sequencing (WES) identified a homozygous variant (c.865G>A, p.Val289Ile) in the gene, responsible for N-Cadherin production, crucial for cell-cell adhesion. Predicted to be likely pathogenic, the variant was found in a patient deficient in , , , and . Its impact on cell adhesion was confirmed in L1 fibroblast cell lines.

OBJECTIVE

Create a knockdown in zebrafish for investigating its role in pituitary development through growth hormone and transcription factors expression.

METHODS

Utilized pET28B-RfxCas13d-His plasmid for Cas13 mRNA production via transcription, guiding Cas13 to with three RNAs. Injected the complex into single-cell embryos for analysis up to 96 hpf. Assessed gene expression of , , , and using RT-qPCR. Evaluated cdh2 protein expression through the western blot technique.

RESULTS

Knockdown animals displayed developmental delay. The expression decreased by 75% within 24 hours, rebounded by 48 hours, and reached wild-type levels by 96 hpf. expression decreased at 48h but increased by 96 hpf, aligning with WT. No significant differences in and expression were observed.

CONCLUSION

Our findings underscore 's role in pituitary development and hormonal regulation, offering insights for developmental biology research.