RESUMO
We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and accounts for genetic variation, which allows allele-specific guide design on a genome-wide scale. The latter ability holds promise for the development of gene therapy in the context of gene dosing and dominant negative mutations.
RESUMO
Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.
Assuntos
Diferenciação Celular , Inflamação , Macrófagos , Monócitos , RNA Longo não Codificante , Transdução de Sinais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/citologia , Diferenciação Celular/genética , Monócitos/metabolismo , Monócitos/citologia , Inflamação/genética , Inflamação/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , NF-kappa B/metabolismo , Transativadores/metabolismo , Transativadores/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Sistemas CRISPR-Cas , Regulação da Expressão GênicaRESUMO
Long noncoding RNAs (LncRNAs) are key regulators of gene expression and can mediate their effects in both the nucleus and cytoplasm. Some of the best-characterized lncRNAs are localized within the nucleus, where they modulate the nuclear architecture and influence gene expression. In this review, we discuss the role of lncRNAs in nuclear architecture in the context of their gene regulatory functions in innate immunity. Here, we discuss various approaches to functionally characterize nuclear-localized lncRNAs and the challenges faced in the field.
Assuntos
Núcleo Celular , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Animais , Imunidade Inata , Regulação da Expressão GênicaRESUMO
Paraoxonase 1(PON1) is an HDL-associated protein, which metabolizes inflammatory, oxidized lipids associated with atherosclerotic plaque development. Because oxidized lipid mediators have also been implicated in the pathogenesis of rheumatoid arthritis (RA), we evaluated the role of PON1 in murine inflammatory arthritis. K/BxN serum transfer (STIA) or collagen antibody transfer (CAIA) was used for arthritis induction in B6 mice homozygous for the PON1 human transgene [PON1Tg], PON1 knock-out mice [PON1KO], and wild type littermate control mice [WT]. Experiments were also performed in K/BxN mice with chronic arthritis, and in RA patients and healthy controls. Arthritis activity in K/BxN mice was associated with a marked dyslipidemia, lower PON1 activity and higher bioactive lipid mediators (BLM), as well as a dysregulated hepatic lipid gene expression profile. Higher serum PON1 activity correlated with lower BLM and lower arthritis activity in both K/BxN mice and RA patients. Overexpression of the human PON1 transgene was associated with reduced inflammatory arthritis, which correlated strongly with higher circulating PON1 activity, upregulation of the hepatic glutathione pathway, and reduction of circulating BLM. These results implicate PON1 as a potential novel therapeutic target for joint disease in RA with potential for vascular benefit, which warrants further investigation.