Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d
- 1Indiana University, Indianapolis
Abstract
Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.
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BibTeX
@misc{kabir2026capindependent,
title = {{Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d}},
author = {M. L. Kabir and S. G. Kodikara and N. AlRamel and J. Alfehaid and P. Pokhrel and S. Basu and H. Balci},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.04.749420},
url = {https://www.biorxiv.org/content/10.64898/2026.09.04.749420v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Cap-Independent Translation Regulation of VEGF Using CRISPR-dCas13d
AU - M. L. Kabir
AU - S. G. Kodikara
AU - N. AlRamel
AU - J. Alfehaid
AU - P. Pokhrel
AU - S. Basu
AU - H. Balci
PY - 2026
DA - 2026/09/04
PB - bioRxiv
DO - 10.64898/2026.09.04.749420
UR - https://www.biorxiv.org/content/10.64898/2026.09.04.749420v1
AB - Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.
ER -