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  • Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates. Nature communications Aris, K. D., Cofsky, J. C., Shi, H., Al-Sayyad, N., Ivanov, I. E., Balaji, A., Doudna, J. A., Bryant, Z. 2025; 16 (1): 2939

    Abstract

    The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5bp initial RNA-DNA hybridization and a ~17bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.

    View details for DOI 10.1038/s41467-025-57703-y

    View details for PubMedID 40133266

  • Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles. Nature communications Andronov, L., Han, M., Zhu, Y., Balaji, A., Roy, A. R., Barentine, A. E., Patel, P., Garhyan, J., Qi, L. S., Moerner, W. E. 2024; 15 (1): 4644

    Abstract

    The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

    View details for DOI 10.1038/s41467-024-48991-x

    View details for PubMedID 38821943

    View details for PubMedCentralID 7951565

  • Revealing the 3D nanoscale organization of MyosinH in the apical complex of toxoplasma gondii through single-molecule localization microscopy with the double-helix point spread function Balaji, A., Zarko, L., Dahlberg, P. D., Boothroyd, J. C., Moerner, W. E. CELL PRESS. 2024: 30A-31A

    View details for Web of Science ID 001194120700146

  • Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles. bioRxiv : the preprint server for biology Andronov, L., Han, M., Zhu, Y., Roy, A. R., Barentine, A. E., Garhyan, J., Qi, L. S., Moerner, W. E. 2023

    Abstract

    The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

    View details for DOI 10.1101/2023.11.07.566110

    View details for PubMedID 37986994

  • Stabilizing a centrifugal force microscope to study DNA folding Orlowski, A. A., Huq, M., Balaji, A., Carter, A. R. CELL PRESS. 2023: 548A

    View details for Web of Science ID 000989629703040

  • Stabilizing a centrifugal force microscope to study DNA folding. Biophysical journal Orlowski, A. A., Huq, M., Balaji, A., Carter, A. R. 2023; 122 (3S1): 548a

    View details for DOI 10.1016/j.bpj.2022.11.2898

    View details for PubMedID 36784841

  • Characterizing the distribution of myosin H in the apical complex of conoid protruded and conoid retracted Toxoplasma gondii Balaji, A., Dahlberg, P. D., Segev-Zarko, L., Sun, S., Chiu, W., Boothroyd, J., Moerner, W. E. CELL PRESS. 2022: 409A

    View details for Web of Science ID 000759523002535

  • Single-molecule analysis of DNA interrogation by Cas9 and Cas12a on supercoiled DNA Aris, K. P., Cofsky, J., Wright, A. V., Ivanov, I. E., Balaji, A., Doudna, J. A., Bryant, Z. CELL PRESS. 2022: 288A

    View details for Web of Science ID 000759523001663

  • Construction of a centrifugal force microscope Golden, G. N., Carter, A., Balaji, A. CELL PRESS. 2022: 417A-418A

    View details for Web of Science ID 000759523002577