Daesun Song

All Publications

• Non-invasive control of gene editing in vivo by photoswitchable Cas12f and focused ultrasound. Cell Song, D., Malinao, M. G., Wu, X., Jiang, S., Jiang, D., Westberg, M., Su, Y., Cai, S., Lee, D., Repina, N. A., Schaffer, D. V., Ji, H. P., Hong, G., Lin, M. Z. 2026

  Abstract

  A spatiotemporally controllable CRISPR-Cas system would be highly desirable for restricting gene editing to specific times and locations in vivo. Ideally, such a system would be compatible with tissue- or cell-type-specific delivery by adeno-associated virus (AAV) and could be controlled from outside the body. However, neither the delivery of controllable CRISPR-Cas systems by single AAV particles nor their noninvasive activation deep within tissue has yet been achieved. In this study, we address both of these limitations. We engineer compact photoswitchable Cas12f (psCas12f) proteins that are co-deliverable with their single guide RNA (sgRNA) in a single AAV particle. We then activate psCas12f in vivo using focused ultrasound (FUS) and mechanoluminescent particles, enabling precise spatiotemporal control. We demonstrate successful gene editing in the targeted skeletal muscle and brain regions of mice using AAV-transduced psCas12f and FUS. In summary, we describe a completely noninvasive method for localized gene editing in vivo.

  View details for DOI 10.1016/j.cell.2026.09.005

  View details for PubMedID 42805175

• Fast analysis and engineering of protein function by microbe-independent deep assembly and screening. Molecular systems biology Wu, Y., Wang, P., Liu, L. X., Song, D., Qin, Q., Gao, C., Hageman, M., Kirkland, T. A., Su, Y., Lin, M. Z. 2026

  Abstract

  Traditional methods for engineering and sequence-fitness analysis of proteins in mammalian cells are limited by the time, cost, and labor associated with plasmid cloning and preparation. Here we present Microbe-Independent Deep Assembly and Screening (MIDAS), a deterministic platform for rapid protein variant expression and characterization in mammalian cells that bypasses microbial cloning by directly transfecting PCR-assembled genes. MIDAS enables high-quality sequence-fitness assessment of arbitrary mutational spaces, including truly deep saturation mutagenesis and combinatorial variant assembly, requiring less than one workday from initial PCR to cell transfection. Using MIDAS, we engineer a high-performance acetylcholine neurotransmitter bioluminescent indicator (ACh-NeuBI), achieving stepwise improvements in responsivity through linker engineering, single-site, and multi-site mutagenesis. We also apply MIDAS to engineer improved NanoLuc luciferase variants for multiple substrates, and to characterize the structural basis of mutational tolerance and substrate specificity. Thus, MIDAS is a versatile method for rapid plasmid-free protein engineering and sequence-fitness analysis in mammalian cells, offering a practical alternative to cloning-based approaches for many protein optimization and characterization tasks.

  View details for DOI 10.1038/s44320-026-00210-z

  View details for PubMedID 42026235

  View details for PubMedCentralID 11168924

• Steric disruption of EGFR oligomerization overcomes therapy resistance in non-small cell lung cancer. Science advances Song, D., Jang, J., Lin, M. Z., Jung, Y. 2025; 11 (49): eady7032

  Abstract

  Acquired drug resistance mutations in epidermal growth factor receptor (EGFR) present a substantial clinical challenge in treating non-small cell lung cancer (NSCLC). While EGFR oligomerization plays a pivotal role in modulating receptor signaling, its relationship with resistance mutations remains unclear. Here, we investigated the real-time link between oligomerization of oncogenic and resistant EGFR mutants and downstream signaling using a fluorescent protein-based proximity probe and signaling reporter in living cells. We found that EGFR mutants resistant to tyrosine kinase inhibitors (TKIs) exhibited higher oligomerization than did wild-type EGFR with or without EGF. The efficacy of TKIs and allosteric drugs inversely correlated with receptor oligomerization. Furthermore, sterically disrupting EGFR oligomerization by genetically fusing a bulky protein to resistant mutants overcame resistance and suppressed proliferation. Moreover, extracellular application of bulky EGFR binders suppressed resistant mutants by disrupting oligomerization. These findings highlight steric disruption of EGFR oligomerization as a promising strategy for overcoming therapy resistance in NSCLC and introduce a versatile screening platform for developing competitive and allosteric inhibitors.

  View details for DOI 10.1126/sciadv.ady7032

  View details for PubMedID 41348890

• Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions NATURE COMMUNICATIONS Song, D., Jo, Y., Choi, J., Jung, Y. 2020; 11 (1): 5642

  Abstract

  Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid-liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

  View details for DOI 10.1038/s41467-020-19476-4

  View details for Web of Science ID 000591592300010

  View details for PubMedID 33159068

  View details for PubMedCentralID PMC7648067

• Behavior control of membrane-less protein liquid condensates with metal ion-induced phase separation NATURE COMMUNICATIONS Hong, K., Song, D., Jung, Y. 2020; 11 (1): 5554

  Abstract

  Phase separation of specific biomolecules into liquid droplet-like condensates is a key mechanism to form membrane-less organelles, which spatio-temporally organize diverse biochemical processes in cells. To investigate the working principles of these biomolecular condensates as dynamic reaction centers, precise control of diverse condensate properties is essential. Here, we design a strategy for metal ion-induced clustering of minimal protein modules to produce liquid protein condensates, the properties of which can be widely varied by simple manipulation of the protein clustering systems. The droplet forming-minimal module contains only a single receptor protein and a binding ligand peptide with a hexahistidine tag for divalent metal ion-mediated clustering. A wide range of protein condensate properties such as droplet forming tendency, droplet morphology, inside protein diffusivity, protein recruitment, and droplet density can be varied by adjusting the nature of receptor/ligand pairs or used metal ions, metal/protein ratios, incubation time, binding motif variation on recruited proteins, and even spacing between receptor/ligand pairs and the hexahistidine tag. We also demonstrate metal-ion-induced protein phase separation in cells. The present phase separation strategy provides highly versatile protein condensates, which will greatly facilitate investigation of molecular and structural codes of droplet-forming proteins and the monitoring of biomolecular behaviors inside diverse protein condensates.

  View details for DOI 10.1038/s41467-020-19391-8

  View details for Web of Science ID 000591843300009

  View details for PubMedID 33144560

  View details for PubMedCentralID PMC7642319

• Homo-molecular Fluorescence Complementation for Direct Visualization of Receptor Oligomerization in Living Cells ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Song, D., Jung, Y. 2019; 58 (7): 2045-2049

  Abstract

  Cell surface receptor oligomerization is an attractive target process for drug screening. However, simple but reliable (and thus high-throughput) visualization methods for receptor oligomerization are still lacking. Herein, we report on a new single-construct homo-molecular fluorescence complementation (Homo-FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal-to-noise ratio was not affected by expression level variations of fused receptors. The Homo-FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo-FC reliably visualized the oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI.

  View details for DOI 10.1002/anie.201812780

  View details for Web of Science ID 000458828000028

  View details for PubMedID 30561874