A novel CRISPR-Cas9 nickase-mediated rolling circle amplification (CRIRCA) technique for gene identification and quantitative analysis of extrachromosomal DNA.

[INTRODUCTION] Extrachromosomal DNA (ecDNA) plays an important role in the initiation and progression of cancerous tumors. Although Circle-seq and other genetic technologies can be utilized for ecDNA analysis, they fail to provide multi-dimensional information from ecDNA, which is time-consuming and laborious.

[OBJECTIVES] Herein, by combining the netlike rolling circle amplification (NRCA) with CRISPR, we developed a novel CRISPR-Cas9 nickase-mediated RCA (CRIRCA) technology that can meet the clinical analysis needs of ecDNA.

[METHODS] Atomic force microscope (AFM) was applied to confirm the circular structure of the ecDNA. Agarose gel electrophoresis was performed to analyze the CRIRCA products. Fluorescent detection was applied to characterize the fluorescence signal of amplified products. qPCR and FISH techniques were applied to verify the CRIRCA results of gene identification of ecDNA.

[RESULTS] Our data revealed that CRIRCA achieved more efficient signal amplification compared to traditional RCA methods, allowing it to sensitively analyze small amounts of ecDNA in single tumor cells. Utilizing computer-aided design, we successfully constructed the primer library and sgRNA library of oncogene in ecDNA, and adopted CRIRCA technology to identify the oncogenes of ecDNA in breast cancer cells.

[CONCLUSION] Therefore, CRIRCA can simultaneously obtain the information from structure, sequence and quantitation of ecDNA. This work will fill the gap in the current research on the early monitoring of cancer targeting ecDNA, and provide support for the accurate diagnosis and treatment of cancer.