A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass.

Xuehui Liu,Xiaowo Wang,Michael Q. Zhang,Liyang Liu,Qiongye Dong,Zhen Xie,Lei Wei

doi:10.18632/aging.102034

Abstract

Cellular senescence is an important mechanism of autonomous tumor suppression, while its consequence such as the senescence-associated secretory phenotype (SASP) may drive tumorigenesis and age-related diseases. Therefore, controlling the cell fate optimally when encountering senescence stress is helpful for anti-cancer or anti-aging treatments. To identify genes essential for senescence establishment or maintenance, we carried out a CRISPR-based screen with a deliberately designed single-guide RNA (sgRNA) library. The library comprised of about 12,000 kinds of sgRNAs targeting 1378 senescence-associated genes selected by integrating the information of literature mining, protein-protein interaction network, and differential gene expression. We successfully detected a dozen gene deficiencies potentially causing senescence bypass, and their phenotypes were further validated with a high true positive rate. RNA-seq analysis showed distinct transcriptome patterns of these bypass cells. Interestingly, in the bypass cells, the expression of SASP genes was maintained or elevated with CHEK2, HAS1, or MDK deficiency; but neutralized with MTOR, CRISPLD2, or MORF4L1 deficiency. Pathways of some age-related neurodegenerative disorders were also downregulated with MTOR, CRISPLD2, or MORF4L1 deficiency. The results demonstrated that disturbing these genes could lead to distinct cell fates as a consequence of senescence bypass, suggesting that they may play essential roles in cellular senescence.

Highlights

Cellular senescence is a cell fate with stable cell cycle arrest triggered by a variety of stimuli, such as telomere attrition, DNA damage, oxidative stress, and oncogene activation [1]
The pooled screen for cellular senescence bypass relies on the alterations of the cell growth rate in bypass cells, which contains more considerable noise compared with screens by cell viability
We selected 1378 genes related to cellular senescence from our Human Cellular Senescence Gene Database (HCSGD) [17] by integrating information of three methods, including literature mining, protein-protein interaction (PPI) network and differential gene expression data from dozens of microarrays

Summary

Introduction

Cellular senescence is a cell fate with stable cell cycle arrest triggered by a variety of stimuli, such as telomere attrition, DNA damage, oxidative stress, and oncogene activation [1]. A number of senescence bypass genes have been unveiled by functional screen methods such as retroviral cDNA libraries [7, 8] and shRNA libraries [9, 10]. These screens usually required isolations and expansions of bypass colonies to amplify the moderate proliferative signatures from a large number of genes in the screen library

Methods

Results

Conclusion