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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

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Rolling circle amplification for next-generation molecular diagnostics, genome analysis, and spatial transcriptome

Jueun Han¹, Yejin Song¹, Jihee Lee¹

¹Department of Bioengineering & Nano-Bioengineering, Research Center for Bio Materials and Process Development, Incheon National University, Incheon 22012, Republic of Korea. e.kim@inu.ac.kr.

|September 8, 2025

Summary

Rolling circle amplification (RCA) is a powerful isothermal DNA technology for sensitive molecular diagnostics and multi-omics research. Advancements enhance its use in disease detection, genome analysis, and spatial transcriptomics.

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Area of Science:

Molecular Biology
Biotechnology
Genomics

Background:

Rolling circle amplification (RCA) is a robust isothermal DNA amplification method.
It offers high sensitivity, specificity, and scalability for molecular diagnostics and multi-omics.
RCA generates long, repetitive DNA sequences with high fidelity.

Purpose of the Study:

To review the principles, modifications, and applications of RCA.
To highlight RCA's role in molecular diagnostics, genome analysis, and spatial transcriptomics.
To discuss challenges and future directions for RCA.

Main Methods:

Exploration of various RCA formats (solution-phase, solid-phase, hydrogel-based, digital).
Integration of RCA with CRISPR, biosensors, and nanoparticle amplification.
Application in extrachromosomal circular DNA sequencing and in situ RNA sequencing.

Main Results:

RCA enables ultrasensitive detection of nucleic acids, proteins, and cells.
It provides insights into genome instability, cancer evolution, and cellular heterogeneity.
RCA facilitates high-resolution gene expression mapping in tissues.

Conclusions:

RCA is a versatile tool transforming molecular diagnostics and biomedical research.
Future integration with high-throughput platforms will advance biomarker discovery and multi-omics.
RCA is poised for a significant role in precision medicine and next-generation sequencing.