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Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR–p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR–p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use.Significance:CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR–p53 interactome, which can be suppressed by transient inhibition of p53.

Supplementary Figure from CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies

Supplementary Table from CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies

Supplementary Data from CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies

Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases. Nanyang Technological University Published version This research was funded by grants from the Karolinska Institutet, Swedish Research Council, Swedish Cancer Society, Stiftelsen Professor Nanna Svartz Fond, the China Scholarship Council, and the Nanyang Technological University–Karolinska Institutet Joint PhD Programme. This work has additionally received support via the European Union/ European Federation of Pharmaceutical Industries and Associations (EU/EFPIA) Innovative Medicines Initiative Joint Undertaking (RTCure Grant 777357).

Puerarin, one of the major isoflavonoid compounds isolated from Gegen, is widely used to treat glucose and lipid disorders. Identifying the mechanism of puerarin’s action against lipid and glucose metabolism dysfunction is very important for its application and development. Based on the structure of puerarin, different chemical probes were synthesized to capture and identify puerarin’s protein target. A membrane translocation assay was conducted to identify the interaction between puerarin and target proteins. The results show that puerarin targets the PH domain of Akt1, inhibits Akt1′s transmembrane effect and activates Akt1. Activated Akt1 improves the phosphorylation or activity of downstream proteins, such as GSK-3β and PDE3, and ultimately regulates glucose and lipid metabolism. Puerarin, as a dietary supplement, could target the PH domain of Akt1 to activate the insulin pathway and improve insulin resistance, thereby assisting in the regulation of glucose and lipid metabolism disorders.

Inactivating

CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1–3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivatingp53mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6–8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment ofp53mutated cells, including strong baselineCDKN1Aexpression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.

CRISPR/Cas9 can be used as an experimental tool to inactivate genes in cells. However, a CRISPR-targeted cell population will not show a uniform genotype of the targeted gene. Instead, a mix of genotypes is generated - from wild type to different forms of insertions and deletions. Such mixed genotypes complicate analyzing the role of the targeted gene in the studied cell population. Here, we present a rapid experimental approach to functionally analyze a CRISPR-targeted cell population that does not involve generating clonal cell lines. As a simple readout, we leverage the CRISPR-induced genetic heterogeneity and use sequencing to identify how different genotypes are enriched or depleted related to the studied cellular behavior or phenotype. The approach uses standard PCR, Sanger sequencing, and a simple sequence deconvoluting software, enabling laboratories without specific in-depth knowledge to also perform these experiments. As proof of principle, we present examples studying the role of different genes for various aspects related to hematopoietic cells (T cell development in vivo and activation in vitro, macrophage phagocytosis, and a leukemia-like phenotype induced by overexpressing a proto-oncogene). In conclusion, we present a rapid experimental approach to identify potential drug targets related to mature immune cells, as well as normal and malignant hematopoiesis.Highlights‐CRISPR generates genetic heterogeneity at the targeted site.‐Genetic heterogeneity complicates identifying the role of a targeted gene.‐Heterogeneity can be quantified by Sanger sequencing with sufficient sensitivity.‐Enrichment of specific genotypes can be used to identify roles for targeted genes.‐Competitive experiments show the potential of genotype enrichment as a discovery tool.Graphical representation

Significance Joint inflammation is the defining characteristic of the autoimmune disease rheumatoid arthritis. Here we use experimental systems to show that the Th2 cytokine IL-4 suppresses the neutrophil potential of causing acute joint inflammation, including suppressing their migration to the inflamed joint, and by upregulating their expression of the inhibitory IgG Fc receptor. We also identify that the pathway is regulated by colony stimulating factor 3. Identifying strategies for early intervention in patients before they develop chronic, debilitating disease is a key focus of the current research in autoimmune diseases. Our study identifies mechanisms that could be exploited therapeutically in patient groups where IgG autoantibodies and neutrophils are thought to play an important role in the pathology., Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4– and IL-13–induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.

Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid widely existing in various medicinal plants, has been reported to have multifarious biological activities such as anti-inflammatory, anticancer and antioxidant activities. In this paper, we analyzed the metabolic profile of UA in mice (including plasma and urine) by using ultra-high performance liquid chromatography (UPLC) coupled with a quadrupole time-of-flight (Q/TOF) method. Principal component analysis (PCA) was applied to differentiate the control and experimental groups. Potential biomarkers were filtered by using loading plots followed by further analysis with UPLC-Q/TOF-MS data. The results showed that 3 metabolites in plasma were identified as markers, one of which was UA and the others were UA epoxides, which belonged to phase I metabolites. Additionally, 5 phase II metabolites were tentatively identified in urine through an accurate mass and characteristic fragment ions. These data suggested that the biotransformation of UA undergoes the major metabolic reactions of the phase I metabolic route of olefin oxidation and phase II metabolic routes of glycine conjugation, glutathione conjugation and glucuronidation. This is the first report of analysis and characterization of the metabolites after the oral administration of UA in mice. The proposed metabolic pathways of UA in mice is also raised for the first time. It might provide further understanding of the potential biological mechanism of UA.