A CRISPRi Screen to Identify RNA Binding Proteins With Oncogenic Functions in Colorectal Cancer

Aug 14, 2024·
Meghan Forsythe
Meghan Forsythe
· 2 min read
Abstract
This project examines RNA Binding Proteins (RBPs) crucial for colorectal cancer cell proliferation, applying a CRISPRi screen in HCT116 cell lines to identify three key regulators: PELO, RBM22, and ZMAT2. The findings indicate that PELO is upregulated in CRC patient tissues and is essential for cell survival, supported by DepMap analysis. Current validation explores targeted knockdown of these proteins and investigates their molecular effects on cancer phenotype.
Type
Publication
NIH Summer Poster Day & Presentation, National Cancer Institute, Bethesda, MD

Project Overview

During the 2024 NIH Summer Internship, research focused on identifying and validating RNA Binding Proteins in colorectal cancer using a genome-wide CRISPRi screen. The top hits—PELO, RBM22, ZMAT2—were found to be critical for cancer cell proliferation and survival, as confirmed by patient expression data and DepMap analyses. These candidates are now being further validated in HCT116 cells by CRISPRi-mediated knockdown and phenotypic assays[attached_file:1][file:150][file:151].

Methods & Results

  • CRISPRi screens in HCT116 cell lines narrowed down candidates to PELO, RBM22, and ZMAT2 after examining gene depletion and enrichment patterns[attached_file:1][file:150][file:151].
  • PELO is upregulated in CRC patients, and its depletion reduces cell proliferation; similar essentiality observed for RBM22 and ZMAT2[attached_file:1][file:150][file:151].
  • Validation includes quantitative PCR, Western Blot confirmation of knockdowns, and functional assays for proliferation, apoptosis, and migration[attached_file:1][file:150][file:151].
  • Ongoing efforts will explore genome-wide regulatory mechanisms impacted by these RBPs and assess broader therapeutic implications[attached_file:1][file:150][file:151].

Author Experience

This project was presented at NIH Poster Day 2024 and selected for oral presentation based on research excellence. The experience provided both technical advancement in CRISPR screening approaches and invaluable mentorship from academic and industry collaborators, strengthening motivation toward a translational cancer research career trajectory[attached_file:1][file:151].

Media

Acknowledgements

Special thanks to Ashish Lal, Ioannis Grammatikakis, Raj Chari, Xiao Ling Li and the Lal group (CCR, NCI, NIH), as well as mentors at UCSB and NIH for support during this project[attached_file:1][file:151].

References

  • Gebauer F, Schwarz T, Valcárcel J, Hentze MW. Nature Reviews Genetics, 2020.
  • Liu J, Cao X. Cell Research, 2023.
  • Mohamad N et al. CRISPRi Journal, 2024.
Colorectal Cancer RNA Binding Proteins CRISPRi
Meghan Forsythe
Authors
Meghan Forsythe
Molecular & Cellular Biology Researcher. UCSB Honors College. PhD applicant.