小柯机器人

美国斯坦福大学Howard Y. Chang团队近期取得重要工作进展，他们研究通过CRISPR-CATCH对人类染色体外DNA进行靶向分析。相关研究成果2022年10月17日在线发表于《自然—遗传学》杂志上。

研究人员利用CRISPR-CATCH、体外CRISPR-Cas9处理和DNA脉冲场凝胶电泳等技术，分离出分离兆碱基大小的人类ecDNA。研究人员发现含有EGFR、FGFR2和MYC的ecDNA分子在人类癌细胞中强烈富集，NRAS ecDNA在获得性治疗抗性的人类转移性黑色素瘤中富集。ecDNA相对于染色体DNA的靶向富集使遗传变异的分阶段成为可能，确定了EGFRvIII突变仅存在于ecDNA上，并支持胶质母细胞瘤模型中ecDNA发生的切除模型。

CRISPR-CATCH和纳米孔测序技术使单分子ecDNA甲基化分析成为可能，并揭示了ecDNA上EGFR启动子的低甲基化。研究人员通过碱基对分辨率的大小和序列来区分同一样本中的异质性ecDNA物种，并发现了功能专门化的ecDNA，它们扩增了选定的增强子或致癌基因编码序列。

据介绍，染色体外DNA（ecDNA）是一种常见的癌基因扩增模式，但ecDNA的分析具有挑战性。

附：英文原文

Title: Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Author: Hung, King L., Luebeck, Jens, Dehkordi, Siavash R., Coln, Caterina I., Li, Rui, Wong, Ivy Tsz-Lo, Coruh, Ceyda, Dharanipragada, Prashanthi, Lomeli, Shirley H., Weiser, Natasha E., Moriceau, Gatien, Zhang, Xiao, Bailey, Chris, Houlahan, Kathleen E., Yang, Wenting, Gonzlez, Roco Chamorro, Swanton, Charles, Curtis, Christina, Jamal-Hanjani, Mariam, Henssen, Anton G., Law, Julie A., Greenleaf, William J., Lo, Roger S., Mischel, Paul S., Bafna, Vineet, Chang, Howard Y.

Issue&Volume: 2022-10-17

Abstract: Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

DOI: 10.1038/s41588-022-01190-0

Source: https://www.nature.com/articles/s41588-022-01190-0

Nature Genetics：《自然—遗传学》，创刊于1992年。隶属于施普林格·自然出版集团，最新IF：41.307
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投稿链接：https://mts-ng.nature.com/cgi-bin/main.plex