Burke, J. N. | bioRxiv Science

bioRxiv2024

Human Anti-Glycan Reactivity is Driven by the Selection of B cells Utilizing Private Antibody Gene Rearrangements that are Affinity Maturated in Germinal Centers

The human antibody repertoire is broadly reactive with carbohydrate antigens represented in the universe of all living things, including both the host/self-as well as the commensal microflora-derived glycomes. Here we have used BCR receptor cloning and expression together with single-cell transcriptomics to analyze the B cell repertoire to the ubiquitous N-acetyl-D-glucosamine (GlcNAc) epitope in human cohorts and dissect the immune phylogeny of this predominant class of antibodies. We find that circulating anti-GlcNAc B cells exhibiting canonical BMem phenotypes emerge rapidly after birth and couple this observation with evidence for germinal center-dependent affinity maturation of carbohydrate-specific B cell receptors in situ during early childhood. Direct analysis of individual B cell clonotypes reveals they exhibit strikingly distinct fine-specificity profiles for palettes of GlcNAc containing moieties. These results suggest that a generalized exposure to complex environmental glycans drives the steady state anti-glycan repertoire.

immunology↗

bioRxiv2018

A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation

Recent developments in CRISPR-based gene editing have provided new avenues to interrogate gene function. However, application of these tools in the central nervous system has been delayed due to difficulties in transgene expression in post-mitotic neurons. Here, we present a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system to drive gene expression in primary neuronal cultures and the adult brain of rodent model systems. We demonstrate robust, modular, and tunable induction of endogenous target genes as well as multiplexed gene regulation necessary for investigation of complex transcriptional programs. CRISPRa targeting unique promoters in the complex multi-transcript gene Brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

neuroscience↗