CASFISH-based method for detecting the expression of chromosomal fragile sites

Chromosomal fragile sites are regions on chromosomes that are prone to breakage. Common fragile sites (CFSs)are a major class of these fragile sites, and their stability is associated with cancer. Traditional methods for detecting the stability of CFSs have many limitations, hindering the advancement of research in this field. To improve the sensitivity and ease of operation of the detection, this study has established a CASFISH-based method for detecting the expression of chromosomal fragile sites. Firstly, single-guide RNAs (sgRNAs)was designed targeting two repetitive sequences on either side of the FRA 16D region of chromosome 16. Then, purified GST-dCas9-EGFP and GST-dCas9-mCherry fusion proteins were utilized to conduct CASFISH detection in U2OS cells. The results indicated that the sgRNA/dCas9 probes could recognize the DNA on both sides of FRA 16D, enabling imaging of FRA 16D within the cells. Under induced replication stress, the dual-color fluorescence probe can also quantitatively detect the breakage of FRA 16D. Further optimization of this approach will facilitate the dynamic observation of CFSs expression in live cells and the study of CFSs stability during DNA replication.