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

aging bypass crispr sasp cellular senescence

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#1 Engadin

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Posted 09 July 2019 - 05:28 PM

From Rejuvenation Science News (RSN)



F U L L   T E X T :   AgING





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 CHEK2HAS1, or MDK deficiency; but neutralized with MTORCRISPLD2, or MORF4L1 deficiency. Pathways of some age-related neurodegenerative disorders were also downregulated with MTORCRISPLD2, 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.





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]. Cellular senescence acts as an important tumor-suppression mechanism, but in the meanwhile, its dark sides may lead to inflammation, tumor promotion, or aging [2]. Senescent cells often exhibit flattened and enlarged morphology, DNA-damage foci and senescence-associated heterochromatin foci (SAHF), as well as altered gene expressions such as the increment of p16INK4a, positive staining for senescence-associated β-galactosidase (SA-β­gal) and senescence-associated secretory phenotype (SASP).


It has been a great challenge to understand the regulatory mechanisms in cellular senescence to control cell fate with its benefits while refraining from its side effects. Senescence processes are well known to be mainly controlled by p53–p21 and/or p16–pRB pathways [34]. Disruptions of genes in these pathways may result in escaping the fate of stable cell cycle arrest when facing senescence-inducing stimuli, or in other words, acquiring the ability to bypass senescence [56]. Identifying genes deficiency of which causes senescence bypass (“senescence bypass genes” for short) helps us gain knowledge about the regulatory process of cellular senescence and tumorigenesis, and thus provides potential targets to control cell fates when encountering senescence stimulus for anti-cancer or anti-aging therapy. A number of senescence bypass genes have been unveiled by functional screen methods such as retroviral cDNA libraries [78] and shRNA libraries [910]. 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.


Recent progress on clustered regularly interspaced short palindrome repeats (CRISPR)-associated nuclease Cas9 system provided novel tools for pooled large-scale screens of gene function [1112]. In these screens, cells are infected with pooled lentiviral single-guide RNA (sgRNA) library, and the abundance of cells carrying functional sgRNAs, which can be detected by deep sequencing, may be amplified or diminished due to the alterations of cell viability [1112], growth rate [13], ability to metastasize [14], or expression of particular biomarkers [15]. Pooled screens by CRISPR-Cas9 knockout libraries show to be more robust, effective and specific compared with traditional screening methods [1116].


In this study, we presented a large-scale pooled CRISPR-Cas9 knockout screen in human primary dermal fibroblasts (BJ) to comprehensively identify genes that are essential for the establishment or the maintenance of senescence. We designed and constructed a CRISPR-Cas9 knockout library targeting genes associated with cellular senescence by integrating the information of literature mining, protein-protein interaction (PPI) network, and differential gene expression data from dozens of microarrays, and performed a pooled screen based on the library. We not only confirmed several known senescence bypass genes but also uncovered a dozen novel gene deficiencies that led to senescence bypass. Further validations of these genes showed a high true positive rate of the screen. RNA-seq analysis of these bypass cells exhibited distinct transcriptome patterns. Bypass cells with MTORCRISPLD2, or MORF4L1 deficiency intriguingly showed a neutralized SASP expression profiles in comparison with senescent cells, and pathways of some age-related neurodegenerative disorders were downregulated in these cells. The results showed that there are distinct consequences of senescence bypass, and implied different roles of these senescence bypass genes in the process of cellular senescence.





A pooled CRISPR-Cas9 knockout screen with a well-designed senescence-associated sgRNA library in human fibroblasts for cellular senescence bypass


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. Therefore, the coverage of cells infected with a certain sgRNA should be increased to detect the moderate alterations of bypass cell abundances precisely. However, it is hard to passage mortal fibroblasts to a vast amount in comparison with cancer cell lines. Besides, cultivating too many cells during a long time from induction to deep senescence (about one month) is costly and labile. Thus, we designed a senescence-associated sgRNA library to enlarge the coverage and to improve the screen performance consequently (Figure 1A). 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, PPI network and differential gene expression data from dozens of microarrays. In addition, 56 negative control genes were also included. A total number of 12,000 sgRNAs were designed using our CRISPR-ERA platform [18] to target these genes (about eight sgRNAs per gene) and were constructed into a pooled lentiviral library (Figure 1A).




Also tagged with one or more of these keywords: aging, bypass, crispr, sasp, cellular senescence

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