A dCas9 proximity reporter for investigating DNA linear and rotational dynamics
4 affiliations
- 1Department of Cell & Developmental Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
- 2Department of Molecular Pharmaceutics, University of Utah, Salt Lake City, Utah, United States of America.
- 3Department of Medicinal Chemistry; Department of Biomedical Engineering, University of Utah, Salt Lake City, Utah, United States of America.
- 4Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, United States of America.
Abstract
The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the β9 or β10 strands of split-NanoLuc® to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment (Δ11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.
Related
- Investigating Ferritin Permeability to Metal-Based Drugs through Molecular Dynamics
- Insights into the Mechanism of Allele Conversion using a Fluorescent Reporter-Based Compound Heterozygous Allele Conversion Reporter (CHACR) Cell Line Model
- Toward Ideal Proximity‐Driven Reactions in Chemical Biology
- Proximity labeling of the Listeria monocytogenes surface reveals pathogen control of a host deubiquitinase
- Editorial Note: Development and validation of a quantitative Proximity Extension Assay instrument with 21 proteins associated with cardiovascular risk (CVD-21)
BibTeX
@article{parvez2026dcas9,
title = {{A dCas9 proximity reporter for investigating DNA linear and rotational dynamics}},
author = {Saba Parvez and Morgan C Marsh and Shawn C Owen and Randall T Peterson},
journal = {PLOS One},
year = {2026},
month = oct,
doi = {10.1371/journal.pone.0355748},
url = {https://doi.org/10.1371/journal.pone.0355748},
note = {CC BY},
}
RIS
TY - JOUR
TI - A dCas9 proximity reporter for investigating DNA linear and rotational dynamics
AU - Saba Parvez
AU - Morgan C Marsh
AU - Shawn C Owen
AU - Randall T Peterson
PY - 2026
DA - 2026/10/29
JO - PLOS One
DO - 10.1371/journal.pone.0355748
UR - https://doi.org/10.1371/journal.pone.0355748
AB - The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the β9 or β10 strands of split-NanoLuc® to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment (Δ11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.
SN - 1932-6203
ER -