Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

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.

染色体外DNA（extrachromosomal DNA, ecDNA）是癌基因扩增的常见模式，但其分析颇具挑战性。本研究提出一种结合体外CRISPR-Cas9处理与琼脂糖包埋基因组DNA脉冲场凝胶电泳的技术，可实现兆碱基级染色体外DNA的靶向纯化（命名为CRISPR-CATCH）。本研究证实，该方法可从人类癌细胞中高效富集携带表皮生长因子受体（EGFR）、成纤维细胞生长因子受体2（FGFR2）以及MYC基因的ecDNA分子。通过靶向纯化ecDNA与染色体DNA，可实现遗传变异的单倍型定相，并在胶质母细胞瘤神经球模型中确凿证实：ecDNA上携带EGFRvIII突变，而染色体DNA上则为野生型EGFR。将CRISPR-CATCH与纳米孔测序相结合，可实现单分子ecDNA甲基化谱分析，并揭示在同一细胞中，ecDNA上的EGFR启动子相较于其内源染色体位点呈现低甲基化状态。最后，通过按分子量分离ecDNA亚型并结合测序，可实现碱基对分辨率下的兆碱基级ecDNA结构精准重构。CRISPR-CATCH为癌症局灶扩增研究的工具库增添了新的手段，将助力探索ecDNA的遗传与表观遗传图谱的相关研究。