CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

The Multifaceted Role of Macrophages in Oncolytic Virotherapy.

One of the cancer hallmarks is immune evasion mediated by the tumour microenvironment (TME). Oncolytic virotherapy is a form of immunotherapy based on the application of oncolytic viruses (OVs) that selectively replicate in and induce the death of tumour cells. Virotherapy confers reciprocal interaction with the host's immune system. The aim of this review is to explore the role of macrophage-mediated responses in oncolytic virotherapy efficacy. The approach was to study current scientific literature in this field in order to give a comprehensive overview of the interactions of OVs and macrophages and their effects on the TME. The innate immune system has a central influence on the TME; tumour-associated macrophages (TAMs) generally have immunosuppressive, tumour-supportive properties. In the context of oncolytic virotherapy, macrophages were initially thought to predominantly contribute to anti-viral responses, impeding viral spread. However, macrophages have now also been found to mediate transport of OV particles and, after TME infiltration, to be subjected to a phenotypic shift that renders them pro-inflammatory and tumour-suppressive. These TAMs can present tumour antigens leading to a systemic, durable, adaptive anti-tumour immune response. After phagocytosis, they can recirculate carrying tissue-derived proteins, which potentially enables the monitoring of OV replication in the TME. Their role in therapeutic efficacy is therefore multifaceted, but based on research applying relevant, immunocompetent tumour models, macrophages are considered to have a central function in anti-cancer activity. These novel insights hold important clinical implications. When optimised, oncolytic virotherapy, mediating multifactorial inhibition of cancer immune evasion, could contribute to improved patient survival.

Diagnostic and analytical performance evaluation of ten commercial assays for detecting SARS-CoV-2 humoral immune response.

OBJECTIVE: Analytical validation of newly released SARS-CoV-2 antibody assays in the clinical laboratory is crucial to ensure sufficient performance in respect to its intended use. We aimed to assess analytical and diagnostic performance of 8 (semi-)quantitative assays detecting anti-nucleocapsid IgG (Euroimmun, Id-Vet) or total Ig (Roche), anti-spike protein IgG (Euroimmun, Theradiag, DiaSorin, Thermo Fisher) or both (Theradiag) and 2 rapid lateral flow assays (LFA) (AAZ-LMB and Theradiag). METHODS: Specificity was evaluated using a cross-reactivity panel of 85 pre-pandemic serum samples. Sensitivity was determined at both the manufacturer's and a 95% specificity cut-off level, using 81 serum samples of patients with a positive rRT-PCR. Sensitivity was determined in function of time post symptoms onset. RESULTS: Specificity for all assays ranged from 92.9% to 100% (Roche and Thermo Fisher) with the exception of the Theradiag IgM LFA (82.4%). Sensitivity in asymptomatic patients ranged between 41.7% and 58.3%. Sensitivity on samples taken <10 days since symptom onset was low (23.3%-66.7%) and increased on samples taken between 10 and 20 days and > 20 days since symptom onset (80%-96% and 92.9%-100%, respectively). From 20 days after symptom onset, the Roche, Id-vet and Thermo Fisher assays all met the sensitivity (>95%) and specificity (>97%) targets determined by the WHO. Antibody signal response was significantly higher in the critically ill patient group. CONCLUSION: Antibody detection can complement rRT-PCR for the diagnosis of COVID-19, especially in the later stage, or in asymptomatic patients for epidemiological purposes. Addition of IgM in LFAs did not improve sensitivity.