Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.
Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.
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DOI：
10.1038/s41588-022-01190-0
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发表时间：
2022-11
期刊：
影响因子：
30.8
通讯作者：
Chang, Howard Y.
中科院分区：
文献类型：
--
作者：
Hung, King L.;Luebeck, Jens;Dehkordi, Siavash R.;Colon, Caterina, I;Li, Rui;Tsz-Lo Wong, Ivy;Coruh, Ceyda;Dharanipragada, Prashanthi;Lomeli, Shirley H.;Weiser, Natasha E.;Moriceau, Gatien;Zhang, Xiao;Bailey, Chris;Houlahan, Kathleen E.;Yang, Wenting;Gonzalez, Rocio 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.
染色体外DNA（ecDNA）是一种常见的癌基因扩增模式，但分析具有挑战性。在这里，我们调整了CRISPR-CATCH，体外CRISPR-Cas9处理和琼脂糖包埋的基因组DNA的脉冲场凝胶电泳，以前为细菌染色体片段开发，以分离兆碱基大小的人类ecDNAs。我们证明了含有来自人类癌细胞的EGFR、FGFR 2和MYC的ecDNA分子以及来自具有获得性治疗耐药性的人类转移性黑色素瘤的NRAS ecDNA的强烈富集。靶向富集ecDNA相对于染色体DNA能够对遗传变异体进行定相，鉴定出仅在ecDNA上存在EGFRvIII突变，并支持胶质母细胞瘤模型中ecDNA发生的切除模型。CRISPR-CATCH随后进行纳米孔测序，能够进行单分子ecDNA甲基化分析，并揭示了ecDNA上EGFR启动子的低甲基化。我们通过碱基对分辨率的大小和序列区分了同一样本中的异质性ecDNA物种，并发现了功能特异性的ecDNA，其扩增选择的增强子或癌基因编码序列。CRISPR-CATCH用于从人类癌细胞中分离含有致癌基因的染色体外DNA（ecDNA）分子。CRISPR-CATCH随后进行纳米孔测序允许进行甲基化分析，突出显示与天然染色体基因座的差异。
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. CRISPR-CATCH is used to isolate extrachromosomal DNA (ecDNA) molecules containing oncogenes from human cancer cells. CRISPR-CATCH followed by nanopore sequencing allows for methylation profiling, highlighting differences from the native chromosomal loci.
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影响因子：
64.5
作者：
Morton, Andrew R.;Dogan-Artun, Nergiz;Scacheri, Peter C.
通讯作者：
Scacheri, Peter C.
影响因子：
5.3
作者：
CARROLL, SM;DEROSE, ML;WAHL, GM
通讯作者：
WAHL, GM
影响因子：
30.8
作者：
Koche, Richard P.;Rodriguez-Fos, Elias;Henssen, Anton G.
通讯作者：
Henssen, Anton G.
影响因子：
7
作者：
Kelly TK;Liu Y;Lay FD;Liang G;Berman BP;Jones PA
通讯作者：
Jones PA
影响因子：
16.6
作者：
Møller HD;Mohiyuddin M;Prada-Luengo I;Sailani MR;Halling JF;Plomgaard P;Maretty L;Hansen AJ;Snyder MP;Pilegaard H;Lam HYK;Regenberg B
通讯作者：
Regenberg B