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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

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.

SUBMITTER: Hung KL

PROVIDER: S-EPMC9649439 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Hung King L KL Luebeck Jens J Dehkordi Siavash R SR Colón Caterina I CI Li Rui R Wong Ivy Tsz-Lo IT Coruh Ceyda C Dharanipragada Prashanthi P Lomeli Shirley H SH Weiser Natasha E NE Moriceau Gatien G Zhang Xiao X Bailey Chris C Houlahan Kathleen E KE Yang Wenting W González Rocío Chamorro RC Swanton Charles C Curtis Christina C Jamal-Hanjani Mariam M Henssen Anton G AG Law Julie A JA Greenleaf William J WJ Lo Roger S RS Mischel Paul S PS Bafna Vineet V Chang Howard Y HY

Nature genetics 20221017 11

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 the ...[more]

PMID: 36253572

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Project description:BackgroundPrimary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer.MethodsCRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells.ResultsAfter efficiently inducing autochthonous liver tumors in mice within 30-60 days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells.ConclusionsSmall eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.

Dataset Information

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Publications

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

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