# Cas9-Leveraged Single-Molecule Characterization of Sparse Plasmid Vectors in Heterogenous DNA Samples

## TL;DR

This paper introduces a new method using Cas9 and fluorescence microscopy to detect and measure rare circular DNA molecules in complex samples without amplification.

## Contribution

A novel single-molecule fluorescence method for characterizing sparse circular DNA using Cas9 and YOYO-1 staining, avoiding amplification or background removal.

## Key findings

- A linear relationship between DNA size and fluorescence intensity was achieved using Cas9 and YOYO-1.
- The method successfully identified distinct subpopulations of rAAV vectors in transduced HEK293 cells.
- The approach enables sensitive analysis of small circular DNA molecules in complex and mass-limited samples.

## Abstract

The detection and characterization of small circular DNA molecules, such as extrachromosomal circular DNA, non-viral episomal vectors, viruses, and plasmids, are crucial in biological and medical research. This study presents a method for identifying and sizing sparse populations of circular DNA in heterogeneous samples using single-molecule fluorescence microscopy. The method leverages the high sequence specificity of Cas9 combined with the fluorescence of YOYO-1-stained DNA to achieve single molecule sensitivity without the need for amplification or background DNA removal. The results show that a linear relationship between DNA size and fluorescence intensity can be achieved with simultaneous co-localization of Cas9 without significantly affecting size estimation. The method was applied to DNA samples from HEK293 cells transduced with recombinant Adeno-Associated Virus (rAAV) vectors. Characterization of DNA samples from the transduced cells revealed distinct subpopulations of viral vectors, highlighting the potential of the method for sensitive analysis of DNA molecules in complex and mass-limited samples. This approach provides a powerful tool for studying the mechanisms of rAAV vector transduction and maintenance, with broader applications in detecting and characterizing small circular DNA molecules in biological and medical research.

The online version contains supplementary material available at 10.1007/s12010-025-05450-7.