The function of SIRT7 in cellular stress response and tissue maintenance

Until the last few decades, aging was thought to be the stochastic accumulation of errors not subject to any active regulation. However, recent evidence suggests that aging is under regulatory control and is subject to modulation by classical signaling pathways. These pathways include the insulin/IGF-1 pathway, the mTOR pathway, and sirtuins, whose up- or downregulation can trigger diverse cell-protective mechanisms against environmental and physiological stress and lead to extension of lifespan.Sirtuins have been shown to play a significant role in tissue maintenance by regulating cellular response to stresses such as genome instability, oxidative stress, and nutritional stress. However, since their role in maintaining proteostasis has not been studied yet, we set out to investigate if SIRT7, a nuclear member of the sirtuin family, functions in regulating protein homeostasis, particularly in the context of protein folding stress. Using a murine model, we focused on studying the function of SIRT7 in the liver and the hematopoietic system, two tissues that SIRT7 is most highly expressed in. We found that in the liver, SIRT7 relieves ER stress through suppressing translation by inhibiting the transcription of ribosomal proteins via Myc. In hematopoietic stem cells, SIRT7 regulates mitochondrial stress by repressing the activity of NRF-1 and inhibiting the transcription of mitochondrial ribosomal subunits. In a physiological context, loss of SIRT7 leads to a steatohepatitis phenotype in the liver and an aging phenotype in the hematopoietic stem cells. Thus, our study suggests the maintenance of stress by SIRT7 as an important mechanism for hepatic lipid metabolism and hematopoietic stem cell function

Molecular, Cellular, and Physiological Characterization of Sirtuin 7 (SIRT7)

Sirtuin 7 (SIRT7), a histone 3 lysine 18 (H3K18) deacetylase, functions at chromatin to suppress endoplasmic reticulum (ER) stress and mitochondrial protein folding stress (PFS(mt)), and prevent the development of fatty liver disease and hematopoietic stem cell aging. In this chapter, we provide a methodology to characterize the molecular, cellular, and physiological functions of SIRT7

In vitro-transcribed guide RNAs trigger an innate immune response via the RIG-I pathway.

Clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro-transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here, we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that is similar to canonical immune-stimulating ligands. IVT gRNAs are recognized in the cytosol through the retinoic acid-inducible gene I (RIG-I) pathway but not the melanoma differentiation-associated gene 5 (MDA5) pathway, thereby triggering a type I interferon response. Removal of the 5'-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells

A mitochondrial UPR-mediated metabolic checkpoint regulates hematopoietic stem cell aging

Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. How stem cells maintain metabolic homeostasis remains elusive. Here, we identified a regulatory branch of the mitochondrial unfolded protein response (UPR(mt)), which is mediated by the interplay of SIRT7 and NRF1 and is coupled to cellular energy metabolism and proliferation. SIRT7 inactivation caused reduced quiescence, increased mitochondrial protein folding stress (PFS(mt)), and compromised regenerative capacity of hematopoietic stem cells (HSCs). SIRT7 expression was reduced in aged HSCs, and SIRT7 up-regulation improved the regenerative capacity of aged HSCs. These findings define the deregulation of a UPR(mt)-mediated metabolic checkpoint as a reversible contributing factor for HSC aging

Transfection of IVT gRNAs induces a cytosolic immune response.

<p>Proposed model of IVT gRNA recognition pathways in mammalian cells. IVT gRNAs carry a 5’-triphosphate, and when complexed with Cas9 protein and transfected into cells, cytosolic RNPs are recognized by RIG-I, triggering a cascade of activation events through the MAVS. This results in phosphorylation of IRF3/7 and their shuttling into the nucleus to activate expression of type I interferons (IFNα/β). This triggers the expression of ISGs. This innate immune response changes the transcriptome of the cell and can cause cell stress and/or death, which in turn might affect the editing outcomes. Cas9, CRISPR-associated 9; gRNA, guide RNA; IFNα/β, interferon alpha/beta; IRF3/7, interferon regulatory factor 3/7; ISG, interferon-stimulated gene; IVT, in vitro–transcribed; MAVS, mitochondrial antiviral signaling protein; RIG-I, retinoic acid–inducible gene I; RNP, ribonucleoprotein.</p

Protospacer and 5’-triphosphate determine the intensity of the gRNA-mediated IFNβ response.

<p>(A) qRT-PCR analysis of <i>IFNB1</i> transcript levels in HEK293 cells transfected with equal amounts of gRNAs containing different 20-nucleotide protospacers. gRNAs were ordered by decreasing levels of <i>IFNB1</i> activation. gRNA1 refers to the gRNA that has been used in all previous experiments. (B) qRT-PCR analysis of <i>ISG15</i> transcript levels in primary HSPCs nucleofected with equal amounts of gRNA 1, 3, 6, 8, and 11 from panel A. Average values of two biological replicates +/−SD are shown. (C) qRT-PCR analysis of <i>IFNB1</i> transcript levels in HEK293 cells transfected with synthetic (“syn”), IVT, and phosphatase-treated IVT gRNAs (gRNA1). (D) Viability of human primary HSPCs 24 h postnucleofection with no RNP and Cas9 or dCas9 RNPs. dCas9 or Cas9 were complexed with synthetic (“syn”) or IVT gRNA targeting the <i>HBB</i> gene. Viability was determined by trypan blue exclusion test. (E) qRT-PCR analysis of <i>ISG15</i> and <i>DDX58</i> (RIG-I) transcript levels in human primary HSPCs 16 h postnucleofection. dCas9 or Cas9 were complexed with synthetic or IVT gRNA targeting the <i>HBB</i> gene, respectively. Ct values were normalized against Ct of mock-nucleofected cells. Average values of two biological replicates +/−SD are shown. (F) Viability of human primary HSPCs 16 h posttransfection with RNPs. RNPs consisted of dCas9 complexed with synthetic, IVT, or CIP-treated IVT gRNAs targeting a noncoding intron of <i>JAK2</i> (left panel) or Cas9 complexed with gRNAs targeting exon 1 of <i>HBB</i> (right panel). Viability was determined by trypan blue exclusion test. (G) Editing outcomes in HSPCs 48 h after nucleofection with RNPs targeting the <i>HBB</i> locus. Indel frequencies were determined by amplicon NGS. Statistical significances were calculated by unpaired <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.0001). The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005840#pbio.2005840.s008" target="_blank">S1 Data</a>. AP, thermosensitive alkaline phosphatase; Cas9, CRISPR-associated 9; CIP, calf intestinal alkaline phosphatase; Ct, cycle threshold; dCas9, nuclease-dead Cas9; <i>DDX58</i>, <i>DExD-H-box helicase 58</i>; gRNA, guide RNA; HEK293, human embryonic kidney 293; HSPC, CD34⁺ human hematopoietic stem and progenitor cell; <i>HBB</i>, <i>hemoglobin subunit beta</i>; IFNβ, interferon beta; <i>IFNB1</i>, <i>interferon beta 1</i>; indel, insertion and deletion; IVT, in vitro–transcribed; <i>JAK2</i>, <i>Janus kinase 2</i>; NGS, next-generation sequencing; n.s., not significant; qRT-PCR, quantitative real-time PCR; PP, 5’ RNA polyphosphatase; RIG-I retinoic acid–inducible gene I; RNP, ribonucleoprotein; SAP, shrimp alkaline phosphatase.</p

SIRT3 Reverses Aging-Associated Degeneration

SummaryDespite recent controversy about their function in some organisms, sirtuins are thought to play evolutionarily conserved roles in lifespan extension. Whether sirtuins can reverse aging-associated degeneration is unknown. Tissue-specific stem cells persist throughout the entire lifespan to repair and maintain tissues, but their self-renewal and differentiation potential become dysregulated with aging. We show that SIRT3, a mammalian sirtuin that regulates the global acetylation landscape of mitochondrial proteins and reduces oxidative stress, is highly enriched in hematopoietic stem cells (HSCs) where it regulates a stress response. SIRT3 is dispensable for HSC maintenance and tissue homeostasis at a young age under homeostatic conditions but is essential under stress or at an old age. Importantly, SIRT3 is suppressed with aging, and SIRT3 upregulation in aged HSCs improves their regenerative capacity. Our study illuminates the plasticity of mitochondrial homeostasis controlling stem cell and tissue maintenance during the aging process and shows that aging-associated degeneration can be reversed by a sirtuin

Transfection of IVT gRNAs into HEK293 cells triggers a type I interferon response.

<p>(A) qRT-PCR analysis of <i>IFNB1</i> and <i>ISG15</i> transcript levels in HEK293 cells transfected with increasing amounts of gRNA with and without Cas9 protein. In the samples with Cas9, gRNAs were complexed with constant amounts (100 pmol, 100 nM final concentration) of Cas9 protein. Cells were harvested for RNA extraction 30 h after transfection using CRISPRMAX transfection reagent. Ct values were normalized to Ct values of mock-transfected HEK293 cells to determine fold activation. (B) qRT-PCR analysis of <i>IFNB1</i> transcript levels in HEK293 cells transfected with equimolar amounts (50 nM) of IVT gRNA, SeV DI RNA, or HCV PAMP, respectively. (C) qRT-PCR analysis of <i>IFNB1</i> and <i>ISG15</i> transcript levels in HEK293 cells over a 48-h time course after transfection with 50 nM via lipofection (Lipofectamine2000 or RNAiMAX) or nucleofection, respectively. For all panels, average values of 3 biological replicates +/−SD are shown. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005840#pbio.2005840.s008" target="_blank">S1 Data</a>. Cas9, CRISPR-associated 9; Ct, cycle threshold; gRNA, guide RNA; HCV, hepatitis C virus; HEK 293, human embryonic kidney 293; <i>IFNB1</i>, <i>interferon beta 1</i>; IVT, in vitro–transcribed; PAMP, pathogen-associated molecular pattern; qRT-PCR, quantitative real-time PCR; SeV DI, Sendai virus defective interfering.</p

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis