首都师范大学学报（自然科学版）2025，Vol.46Issue(5)：47-55,9.DOI:10.19789/j.1004-9398.2025.05.007
基于CASFISH的检测染色体脆性位点表达的方法
CASFISH-based method for detecting the expression of chromosomal fragile sites
摘要
Abstract
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 FRA16D 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 FRA16D,enabling imaging of FRA16D within the cells.Under induced replication stress,the dual-color fluorescence probe can also quantitatively detect the breakage of FRA16D.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.关键词
染色体脆性位点/CFSs/CRISPR/dCas9介导的荧光原位杂交Key words
chromosomal fragile sites/CFSs/CASFISH分类
生物科学引用本文复制引用
张鋆坤,杨雨蒙,吴彤彤,仲昭华,林清雅,吴迪,陈志玲,王海龙,王丹..基于CASFISH的检测染色体脆性位点表达的方法[J].首都师范大学学报（自然科学版）,2025,46(5):47-55,9.基金项目
国家自然科学基金项目(32371354) （32371354）
北京市教委-市自然基金委联合项目(23JE0003) （23JE0003）
