Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

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

doi:10.1038/s41588-022-01190-0

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Nat Genet. 2022 Nov;54(11):1746-1754. doi: 10.1038/s41588-022-01190-0. Epub 2022 Oct 17.

Authors

King L Hung¹, Jens Luebeck^#^{2

3}, Siavash R Dehkordi^#³, Caterina I Colón^{1

4

5}, Rui Li¹, Ivy Tsz-Lo Wong^{4

5}, Ceyda Coruh⁶, Prashanthi Dharanipragada⁷, Shirley H Lomeli⁷, Natasha E Weiser^{1

5}, Gatien Moriceau⁷, Xiao Zhang⁷, Chris Bailey⁸, Kathleen E Houlahan^{9

10

11}, Wenting Yang^{9

10

11}, Rocío Chamorro González¹², Charles Swanton^{8

13

14}, Christina Curtis^{9

10

11}, Mariam Jamal-Hanjani^{13

14}, Anton G Henssen^{12

15

16

17}, Julie A Law⁶, William J Greenleaf^{1

10}, Roger S Lo^{7

18

19}, Paul S Mischel^{4

5}, Vineet Bafna³, Howard Y Chang^{20

21

22}

Affiliations

¹ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA.
² Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, La Jolla, CA, USA.
³ Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA, USA.
⁴ Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
⁵ Department of Pathology, Stanford University, Stanford, CA, USA.
⁶ Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA.
⁷ Division of Dermatology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
⁸ Cancer Evolution and Genome Instability Laboratory, The Francis Crick Institute, London, UK.
⁹ Department of Medicine, Division of Oncology, Stanford University School of Medicine, Stanford, CA, USA.
¹⁰ Department of Genetics, Stanford University, Stanford, CA, USA.
¹¹ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
¹² Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany.
¹³ Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, University College London, London, UK.
¹⁴ University College London Hospitals NHS Trust, London, UK.
¹⁵ Experimental and Clinical Research Center (ECRC), Max Delbrück Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany.
¹⁶ German Cancer Consortium (DKTK), partner site Berlin, and German Cancer Research Center DKFZ, Heidelberg, Germany.
¹⁷ Berlin Institute of Health, Berlin, Germany.
¹⁸ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
¹⁹ Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
²⁰ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, USA. howchang@stanford.edu.
²¹ Department of Genetics, Stanford University, Stanford, CA, USA. howchang@stanford.edu.
²² Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. howchang@stanford.edu.

^# Contributed equally.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

DNA / genetics
ErbB Receptors / genetics
Glioblastoma* / genetics
Humans
Neoplasms* / genetics
Neoplasms* / pathology
Oncogenes

Substances

DNA
ErbB Receptors

Abstract

Publication types

MeSH terms

Substances

Grants and funding