CRISPR-CATCH Improves Human ecDNA Profiling in Tumor Tissue

Extrachromosomal DNA (ecDNA) frequently contributes to oncogene amplification, with current techniques for ecDNA isolation and targeted profiling having limitations. Hung and colleagues sought to improve upon these previous methods by adapting CRISPR-CATCH (Cas9-assisted targeting of chromosome segments), previously developed for bacterial chromosome segments, to enrich for megabase-sized ecDNA. This single cut technique was found to successfully isolate megabase-sized ecDNA and the corresponding chromosomal locus from the same cancer cell sample as well as from archived patient tissues, with a 30-fold enrichment of target ecDNA being observed, which was demonstrated in EGFR, FGFR2, MYC, and NRAS ecDNA. Additionally, the EGFR locus in human glioblastoma cells was used to identify structural variants from short-read sequencing data, which revealed predominance of the EGFRvIII mutation on ecDNA, while full-length EGFR was seen on the chromosomal locus, suggesting ecDNA can harbor unique genetic alterations. Moreover, single-nucleotide variants were significantly divergent on ecDNA compared to chromosomal DNA, which indicates origination from different parental alleles and together contributes to the evidence in support of an excision model of ecDNA genesis. Examination of the feasibility of analyzing epigenomic profiles using this technique, specifically through investigation of DNA cytosine methylation profiles of the EGFR chromosomal locus, demonstrated reduced DNA methylation at regulatory elements on ecDNA, which supports that altered gene regulation can be revealed using CRISPR-CATCH. Furthermore, CRISPR-CATCH was used to directly estimate and reconstruct molecule size and amplicon-phased structural information, establishing the utility of these data to ascertain and provide insight into ecDNA structural and regulatory landscapes. In conclusion, the results of this study show that ecDNA isolated from tumor cells using this CRISPR-CATCH method can be analyzed for both genomic and epigenomic features that will, in turn, allow for greater insight into ecDNA origin, structure, diversity, and regulation in cancer.

Hung KL, Luebeck J, Dehkordi SR, Colón CI, Li R, Wong IT, et al. Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH. Nat Genet 2022 Oct 17 [Epub ahead of print].

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