FACS-Based Genome-Wide CRISPR Screens Define Key Regulators of DNA Damage Signaling Pathways

Authors

Min Huang
Fuwen Yao
Litong Nie
Chao Wang
Dan Su
Huimin Zhang
Siting Li
Mengfan Tang
Xu Feng
Bin Yu
Zhen Chen
Shimin Wang
Ling Yin
Lisha Mou
Traver Hart
Junjie Chen

Publication Date

8-3-2023

Journal

Molecular Cell

Abstract

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

Keywords

Humans, Clustered Regularly Interspaced Short Palindromic Repeats, Flow Cytometry, DNA Damage, Signal Transduction, Ataxia Telangiectasia Mutated Proteins, Genome, Protein-Arginine N-Methyltransferases, Repressor Proteins

Comments

Supplementary Materials

PMID: 37541219