| Literature DB >> 36253572 |
Jens Luebeck1,2, Siavash R Dehkordi2, King L Hung3, Caterina I Colón3,4,5, Rui Li3, Ivy Tsz-Lo Wong4,5, Ceyda Coruh6, Prashanthi Dharanipragada7, Shirley H Lomeli7, Natasha E Weiser3,5, Gatien Moriceau7, Xiao Zhang7, Chris Bailey8, Kathleen E Houlahan9,10,11, Wenting Yang9,10,11, Rocío Chamorro González12, Charles Swanton8,13,14, Christina Curtis9,10,11, Mariam Jamal-Hanjani13,14, Anton G Henssen12,15,16,17, Julie A Law6, William J Greenleaf3,10, Roger S Lo7,18,19, Paul S Mischel4,5, Vineet Bafna2, Howard Y Chang20,21,22.
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.Entities:
Year: 2022 PMID： 36253572 DOI： 10.1038/s41588-022-01190-0
Source DB: PubMed Journal: Nat Genet ISSN： 1061-4036 Impact factor: 41.307