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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

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

in 631/1647/2230/1452 / 631/337/176 / 631/67/395 / Agriculture / Amplification / Animal Genetics and Genomics / Biomedical and Life Sciences / Biomedicine / Brain cancer / Cancer / Cancer Research / Chromosomes / CRISPR / Deoxyribonucleic acid / DNA / DNA - genetics / DNA fingerprinting / DNA methylation / Drug resistance / Electrophoresis / Enhancers / Epidermal growth factor receptors / Epigenetics / ErbB Receptors - genetics / Fibroblast growth factor receptor 2 / Gel electrophoresis / Gene Function / Genetic diversity / Genetic variance / Genomes / Glioblastoma / Glioblastoma - genetics / Human Genetics / Humans / Melanoma / Metastases / Metastasis / Mutation / Myc protein / Neoplasms - genetics / Neoplasms - pathology / Oncogenes / Target acquisition / technical-report

2022

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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

2022

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2022

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Journal Article

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Wong, Ivy Tsz-Lo,

Law, Julie A.,

Yang, Wenting,

Mischel, Paul S.,

Coruh, Ceyda,

Curtis, Christina,

Lomeli, Shirley H.,

Bailey, Chris,

Henssen, Anton G.,

González, Rocío Chamorro,

Weiser, Natasha E.,

Luebeck, Jens,

Dharanipragada, Prashanthi,

Greenleaf, William J.,

Houlahan, Kathleen E.,

Moriceau, Gatien,

Swanton, Charles,

Bafna, Vineet,

Zhang, Xiao,

Hung, King L.,

Lo, Roger S.,

Li, Rui,

Chang, Howard Y.,

Dehkordi, Siavash R.,

Colón, Caterina I.,

Jamal-Hanjani, Mariam

2022

Overview

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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Publisher

Nature Publishing Group US,Nature Publishing Group

Subject

/ Animal Genetics and Genomics

/ Biomedical and Life Sciences