Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

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

TLDR

  • The study is about a new way to find and study a type of DNA called extrachromosomal DNA (ecDNA) that is commonly found in cancer cells. The study uses a new method called CRISPR-CATCH to isolate ecDNA molecules that contain specific genes like EGFR, FGFR2, MYC, and NRAS. The study also finds that ecDNA molecules have different versions of these genes that are not found in the regular DNA. The study also finds that ecDNA molecules have different sizes and sequences that are not found in the regular DNA. The study has important implications for understanding how cancer cells work and how we can treat them.

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.

Overview

  • The study aims to adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment, and pulsed field gel electrophoresis of agarose-entrapped genomic DNA to isolate megabase-sized human extrachromosomal DNA (ecDNA).
  • The study focuses on isolating ecDNA molecules containing EGFR, FGFR2, MYC, and NRAS from human cancer cells and metastatic melanoma with acquired therapeutic resistance. The study also explores the phasing of genetic variants, the presence of an EGFRvIII mutation exclusively on ecDNAs, and an excision model of ecDNA genesis in a glioblastoma model. Additionally, the study uses CRISPR-CATCH followed by nanopore sequencing to enable single-molecule ecDNA methylation profiling and reveals hypomethylation of the EGFR promoter on ecDNAs. The study distinguishes heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovers functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Comparative Analysis & Findings

  • The study demonstrates strong enrichment of ecDNA molecules containing EGFR, FGFR2, MYC, and NRAS from human cancer cells and 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. The study distinguishes heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovers functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Implications and Future Directions

  • The study's findings have significant implications for the field of research and clinical practice, as they provide a novel approach to isolate and analyze ecDNA molecules. The study's findings also have the potential to inform the development of new therapeutic strategies for cancer treatment. Future research directions could include the use of CRISPR-CATCH to isolate ecDNA molecules from other types of cancer cells and the exploration of the role of ecDNA in cancer progression and drug resistance.
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