Work

CRISPR-Cas9 mediated d3GHR knockout in HEK293 cells: Revealing the longevity associated isoform stress resilience

Year: 2024

Type: article

Abstract: The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the “d3GHR,” which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT ( p < 0.05). This noteworthy finding might offer a practical approach to test illness prevention and give the scientific community critical new insights on mechanism associated with lifespan. • We utilized CRISPR/Cas9 to produce homozygous and heterozygous isoforms for deleting GHR gene exon 3 in HEK293 cells. • Homozygous d3GHR cells exhibited significantly lower baseline expression of IGF1 and JAK/STAT genes compared to WT. • Heterozygous d3GHR cells showed higher survival rates under UV radiation and better viability during fasting. • Findings suggest the d3GHR isoform may boost stress resilience and longevity, offering insights into lifespan mechanisms. • Findings offer potential strategies for disease prevention and a better understanding of genetic influences on lifespan.

Source: Experimental Gerontology

DOI: 10.1016/j.exger.2024.112586

Authors: Ghadeer Falah, Lital Sharvit, Gil Atzmon

Institutions: University of Haifa, Albert Einstein College of Medicine

Language: English

Topic: Growth Hormone and Insulin-like Growth Factors

Subfield: Endocrinology, Diabetes and Metabolism

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $2,990