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 present a method for targeted purification of megabase-sized ecDNA by combining in-vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA (CRISPR-CATCH). We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells. Targeted purification of ecDNA versus chromosomal DNA enabled phasing of genetic variants and provided definitive proof of an EGFRvIII mutation on ecDNA and wild-type EGFR on chromosomal DNA in a glioblastoma neurosphere model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNA compared to the native chromosomal locus in the same cells. Finally, separation of ecDNA species by size and sequencing allowed accurate reconstruction of megabase- sized ecDNA structures with base-pair resolution. CRISPR-CATCH is a new addition to the toolkit for studying focal amplifications in cancer and will accelerate studies aiming to explore the genetic and epigenetic landscapes of ecDNA.

iSCORED: nanopore-based random genomic sampling for intraoperative molecular diagnosis

“…While large 600kb bins are effective in detecting aneuploidy, most clinically-relevant gene amplifications occur within a range of hundreds of kilobases to a few megabases 27,28 . In such cases, using a large bin could result in averaging out the dose change, leading to decreased accuracy of small amplifications.…”

“…2c and Figure S9). EGFR amplification is a molecular defining alterations in glioblastomas, typically occurring as extrachromosomal DNA ranging from 1-3 megabases (Mb) in size 27,32 . In our cohort of six EGFR-amplified glioblastomas, the average amplification regions spanned 1.66 ± 0.44 Mb (SEM) with an average copy number of 150.5 ± 47 (SEM).…”

“…The epigenetic landscapes of amplified oncogenes offer mechanistic insights into transcriptional regulations 27,38 . Despite the inherent low-pass nature of iSCORED platform, gene amplification ensures sufficient coverage for methylation profiling in the defined regions.…”

Single‐cell third‐generation sequencing‐based multi‐omics uncovers gene expression changes governed by ecDNA and structural variants in cancer cells

“…The TGS platform nanopore has been used in ecDNA sequencing, both at the bulk level, 18 and after isolation with a novel targeted ecDNA purification method, CRISPR-CATCH. 46 In addition, TGS-based scWGS methods have recently been developed that permit the identification of SVs and ecDNA. 47 Single-cell multi-omics technologies have enabled the integration of information from multiple sequencing modalities, and have facilitated the establishment of a more accurate correlation between genotype (genomic sequence, DNA methylome and chromatin accessibility) and phenotype (transcriptome and proteome).…”

“…Conversely, the long reads obtained from the TGS platform serve as a promising feature that enables the identification of SVs and ecDNA. The TGS platform nanopore has been used in ecDNA sequencing, both at the bulk level, 18 and after isolation with a novel targeted ecDNA purification method, CRISPR‐CATCH 46 . In addition, TGS‐based scWGS methods have recently been developed that permit the identification of SVs and ecDNA 47 …”