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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

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Item Type:Article
Title:Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH
Creators Name:Hung, K.L. and Luebeck, J. and Dehkordi, S.R. and Colón, C.I. and Li, R. and Wong, I.T.L. and Coruh, C. and Dharanipragada, P. and Lomeli, S.H. and Weiser, N.E. and Moriceau, G. and Zhang, X. and Bailey, C. and Houlahan, K.E. and Yang, W. and González, R.C. and Swanton, C. and Curtis, C. and Jamal-Hanjani, M. and Henssen, A.G. and Law, J.A. and Greenleaf, W.J. and Lo, R.S. and Mischel, P.S. and Bafna, V. and Chang, H.Y.
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.
Keywords:DNA, ErbB Receptors, Glioblastoma, Neoplasms, Oncogenes
Source:Nature Genetics
ISSN:1061-4036
Publisher:Nature Publishing Group
Volume:54
Number:11
Page Range:1746–1754
Date:November 2022
Official Publication:https://doi.org/10.1038/s41588-022-01190-0
PubMed:View item in PubMed

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