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Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.

ABSTRACT: Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.

SUBMITTER: Wertz MH

PROVIDER: S-EPMC7181458 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background & aimsLong noncoding RNAs (lncRNAs) are found to have profound impacts on diverse cellular processes. Although high-throughput sequencing studies have shown the differential lncRNA expression profiles between hepatocellular carcinoma (HCC) and nontumor livers, the functional impacts of lncRNAs on HCC development await further investigation. Herein, we sought to address the functional roles of lncRNAs in HCC pathogenesis by in vivo functional screening.MethodsWe performed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/dead CRISPR-associated protein 9 (dCas9) lncRNA activation screening in HCC xenografts. We characterized the clinical relevance of positively selected lncRNAs using transcriptomic data sets. We used CRISPR-based gene activation and knockdown approaches to show the functional roles of positively selected lncRNAs including Cancer Susceptibility 11 (CASC11) in HCC. RNA sequencing and chromatin isolation by RNA purification sequencing were used to investigate the molecular mechanisms of CASC11 in HCC progression.ResultsThe in vivo functional screening identified 1603 positively selected lncRNAs, 538 of which were overexpressed in HCC patients. Systematic transcriptomic data analysis and clinical investigation showed that patients with high expression of these lncRNA candidates correlated with aggressive tumor behaviors. Overexpression of these lncRNAs aggravated HCC cell growth. Detailed characterization of a lncRNA candidate, CASC11, showed its pivotal role in cell proliferation and tumor growth. Mechanistically, chromatin isolation by RNA purification sequencing showed that CASC11 was bound to the CASC11/MYC proto-oncogene shared promoter region on chromosome 8q24. CASC11 modulated the transcriptional activity of MYC in a cis-regulatory manner, which affected the expression of MYC downstream target genes, consequently promoting G1/S progression.ConclusionsOur study showed the power of in vivo CRISPR screening, which comprehensively investigated the functionality of lncRNAs in HCC progression, providing a rationale for targeting these lncRNAs clinically.

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Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.

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