Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA-binding proteins either rely on crosslinking-mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture e ciency. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of speci c lncRNAs in their native cellular context followed by LC-MS/MS identi cation. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.

Mesenchymal stem cells (MSCs) have been increasingly used for treating autoimmune diseases due to their immune modulation functions, but inefficient homing to the target tissue and safety issues limits their wide application. Recently, increasing studies demonstrate small extracellular vesicles (sEVs) as key mediators of MSCs to exert their immunomodulatory effects. In this study, we found that sEVs derived from human umbilical cord MSCs stimulated by IFN-γ (IFNγ-sEVs) inhibited proliferation and activation of peripheral blood mononuclear cells and T cells in vitro. Furthermore, we confirmed that IFNγ-sEVs reduced psoriasis symptoms including thickness, erythema, and scales of skin lesions; exhausted Th17 cells, increased Th2 cells; and reduced enrichment of inflammatory cytokines such as IL-17A, IFN-γ, IL-6, and TNF-α in both spleen and skin lesions in vivo. Importantly, IFNγ-sEVs significantly improved the delivery efficiency and stability of ASO-210, the antisense oligonucleotides of miR-210 block the immune imbalance and subsequent psoriasis development. Our results reveal MSC-sEVs as promising cell-free therapeutic agents and ideal delivery vehicles of antisense oligonucleotides for psoriasis treatment.

Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA-binding proteins either rely on crosslinking-mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of specific lncRNAs in their native cellular context followed by LC-MS/MS identification. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.

PTEN (phosphatase and tensin homologue) is the first tumor suppressor identified to have phosphatase activity and its gene is the second most frequently deleted or mutated tumor-suppressor gene associated with human cancers. Germline PTEN mutations are the cause of three inherited autosomal dominant disorders. Phosphatidylinositol 3,4,5,-triphosphate (PIP3), the product of the PI3 kinase, is one of the key intracellular targets of PTEN's phosphatase activity, although PTEN's phosphatase-independent activities have also been identified. PTEN is critical for stem cell maintenance, which contributes to its controlled tumorigenesis. PTEN loss leads the development of cancer stem cells (CSCs) that share properties with somatic stem cells, including the capacity for self-renewal and multi-lineage differentiation. Methods to isolate and functionally test stem cells and CSCs are important for understanding PTEN functions and the development of therapeutic approaches to target CSCs without having adverse effects on normal stem cells. Here, we describe protocols for the isolation and functional analysis of PTEN deficient embryonic stem cells, hematopoietic stem cells and leukemia-initiating cells (LICs), neural stem cells, and prostate stem cells and CSCs.