Structure and genome editing of type I-B CRISPR-Cas.

Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

Intratumor expanded T cell clones can be non-sentinel lymph node derived in breast cancer revealed by single-cell immune profiling.

BACKGROUND: Sentinel lymph nodes (LNs) are regarded as key immune surveillance sites in cancer wherein mature dendritic cells present tumor-derived antigens to prime and activate T cells, which then migrate to the tumor site. However, it is unclear whether the tumor-specific T cells can be elicited within the tumor independent of the sentinel LNs. METHODS: We performed an integrative analysis of gene expression profiles of 65,285 cells and T cell receptor sequences of 15,831 T cells from 5 paired primary breast tumors and sentinel LNs to identify where clonal T cells come from and the characteristics of those clonal T cells. RESULTS: The proportion of clonal T cells was higher in the primary tumors compared with the sentinel LNs, whereas all expanded clones identified in the sentinel LN were also present in the primary tumors. In contrast, 10.91% of the expanded clones in the primary tumors were not found in the sentinel LNs. These novel intratumoral T cell clones were characterized by high tissues retention capacity (CXCR6 +ITGAE+) and a distinct coinhibitory pattern (CD39 +NKG2A+) compared with the expanded T cell clones common to both sites. Furthermore, multiplex immunofluorescence imaging showed the presence of tertiary lymphoid structures (TLS) in the primary breast tumors wherein the activated cytolytic T cells were concentrated, indicating its possible role in eliciting non-sentinel LN-derived T cell clones. CONCLUSIONS: Our study revealed expanded intratumor non-sentinel LN derived T cell clones located in the TLS, which points to the need for exploring the role of TLS in antitumor immunity.

Integrating genome-wide CRISPR immune screen with multi-omic clinical data reveals distinct classes of tumor intrinsic immune regulators.

BACKGROUND: Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer. METHODS: To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing. RESULTS: Our studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration. CONCLUSIONS: Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations.

Analysis of AgoshRNA maturation and loading into Ago2.

The RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that-after Ago2 cleavage-AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA in vitro. In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects.

Effects of Maillard reaction on allergenicity of buckwheat allergen Fag t 3 during thermal processing.

BACKGROUND: Fag t 3 is a major allergenic protein in tartary buckwheat. The Maillard reaction commonly occurs in food processing, but few studies have been conducted on the influence of thermal processing on the allergenic potential of buckwheat allergen. The aim of the present study was to investigate the effects of autologous plant polysaccharides on the immunoreactivity of buckwheat Fag t 3 (11S globulin) following the Maillard reaction. RESULTS: Fag t 3 and crude polysaccharides were prepared from tartary buckwheat (Fagopyrum tataricum) flour. After heating, the polysaccharides were covalently linked to Fag t 3 via a Maillard reaction, and the IgE/IgG-binding properties of Fag t 3 decreased dramatically, with significant changes also being observed in the electrophoretic mobility, secondary structure and solubility of the glycated Fag t 3. The great influence of glycation on IgE/IgG binding to Fag t 3 was correlated with a significant change in the structure and epitopes of the allergenic protein. These data indicated that conjugation of polysaccharides to Fag t 3 markedly reduced the allergen's immunoreactivity. CONCLUSION: Glycation that occurs via the Maillard reaction during the processing of buckwheat food may be an efficient method to reduce Fag t 3 allergenicity.

Synthesis of hypoallergenic derivatives of the major allergen Fag t 1 from tartary buckwheat via sequence restructuring.

Fag t 1, a legumin-type protein, is the major allergen in tartary buckwheat. In the current study, three recombinant derivatives of Fag t 1, designated as Fag t 1-rs1, Fag t 1-rs2, and Fag t 1-rs3, were constructed via rational design and genetic engineering. However, because of the loss of their native-like folds, the Fag t 1 derivatives failed to bind IgE, and their allergenic activities were reduced. The recombinant hypoallergenic variants are promising vaccine candidates for specific immunotherapy of buckwheat allergy. The unfolding of the Fag t 1 structure reduced its high resistance to gastrointestinal proteolysis and strongly reduced its IgE reactivity. The derivatives showed a more than 90% reduction in allergenic activity compared with rFag t 1. These results suggest that the structure-dependent stability of 11S seed storage proteins is directly related to digestive stability and allergenic potential. Therefore, the destruction of the native conformation is the appropriate strategy to reduce the allergenicity of the cupin family food allergens.