Biology subjects

Southern, N. T.

Publications and source records attributed to Southern, N. T..

3 recordsLinked to original sources

bioRxiv2026

Domain Insertion Improves the Precision of a CRISPR Adenine Base Editor

Adenine base editors (ABEs) enable efficient A*T to G*C conversion, but their broad activity windows frequently cause unintended bystander edits. We hypothesized that insertion of a bulky, inert protein domain into the base editor would limit the effective reach of the deaminase, thereby preferentially directing editing to the intended target adenine. Here, we systematically map domain insertion sites within the high-activity TadA8e adenine base editor using structure-guided design and computational inference. We find that TadA8e accepts domain insertions at multiple surface sites, with overall activity and editing window width dependent on insert position rather than domain identity. Excitingly, inserting domains at residue L68 preserved robust editing across multiple genomic loci while tightly focusing the editing window around position 5. Insertion of superfolder GFP at this site produced a base editor variant with a narrower editing window, the ability to track edited cells by fluorescence, and markedly reduced Cas-independent off-target editing. Our work highlights domain insertion engineering as a powerful strategy to create more focused and precise CRISPR base editors.

synthetic biology↗

bioRxiv2025

Phage-Assisted Evolution of Allosteric Protein Switches

Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation1. Artificial allosteric effectors enable remote manipulation of cell function2,3; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution4 platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering5 to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning [~]1,000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we found that linker mutations promoting -helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerged consistently across independently evolved pools, underscoring their functional relevance. Together, we developed a framework for the directed evolution of programmable allosteric switches in vivo. By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.

synthetic biology↗

bioRxiv2020

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

neuroscience↗