一种用于CRISPR介导的染色体重排的通用报告系统。
A versatile reporter system for CRISPR-mediated chromosomal rearrangements.
作者信息
Li Yingxiang, Park Angela I, Mou Haiwei, Colpan Cansu, Bizhanova Aizhan, Akama-Garren Elliot, Joshi Nik, Hendrickson Eric A, Feldser David, Yin Hao, Anderson Daniel G, Jacks Tyler, Weng Zhiping, Xue Wen
机构信息
Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P. R. China.
RNA Therapeutics Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
出版信息
Genome Biol. 2015 May 28;16(1):111. doi: 10.1186/s13059-015-0680-7.
Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.
虽然染色体缺失和倒位在癌症中很重要，但检测DNA重排的传统方法需要费力的间接检测。在这里，我们开发了荧光报告基因来快速定量CRISPR/Cas9介导的缺失和倒位。我们发现倒位依赖于非同源末端连接酶LIG4。我们还对小鼠肝脏中一个50 kb的Pten基因组区域进行了缺失和倒位工程改造。我们在重排融合位点发现了多样但序列特异的插入缺失。此外，我们检测到非互补链上第四个核苷酸处的Cas9切割，导致产生交错而非平端的DNA断裂。这些报告基因有助于研究染色体重排的机制。
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