原发性肝癌具有显着的肿瘤内遗传异质性（IGH），它推动了癌症的进化并阻碍了有效的癌症治疗。 CRISPR/Cas9 诱导的小鼠肝癌模型可用于阐明 IGH 是如何形成的。然而，由于除了靶向突变之外，CRISPR/Cas9还可以在细胞中诱导染色体碎裂和染色体外DNA，我们想知道这种效应是否有助于CRISPR/Cas9诱导的小鼠肝癌中IGH的发展。基于CRISPR/Cas9的靶向体细胞多重-诱变用于靶向 34 个肿瘤抑制基因 (TSG)，以诱导小鼠原发性肝肿瘤。分析并比较单细胞克隆及其亚克隆之间、细胞增殖的不同时间点之间以及亲本克隆与小鼠皮下同种异体移植物衍生的单细胞克隆之间的肿瘤细胞靶位点突变。基因组不稳定性和染色体外环状 DNA (eccDNA) 的产生被探索为这些肝肿瘤细胞中靶位点突变振荡的潜在机制。在 30-60 天内有效诱导小鼠本土肝肿瘤后，对 CRISPR/Cas9- 的分析诱导的肿瘤和源自肿瘤结节的单细胞克隆揭示了靶位点的多重和异质突变。许多靶位点在单细胞克隆中经常表现出两种以上类型的等位基因变异，且频率不同，表明这些靶位点的拷贝数增加。在单细胞克隆及其亚克隆之间的某些靶位点，靶向 TSG 突变的类型和频率持续发生变化。在没有持续 CRISPR/Cas9 基因组编辑的情况下，即使是细胞培养物和小鼠皮下移植物中亚克隆的增殖也改变了靶向 TSG 突变的类型和频率，这表明在这些肝脏肿瘤中 IGH 的发展存在初级染色体之外的新来源。肿瘤细胞的核型分析揭示了这些细胞中的基因组不稳定性，表现为高水平的微核和染色体畸变，包括染色体片段和染色体断裂。测序分析进一步证明了这些肿瘤细胞中含有靶向 TSG 突变的 eccDNA 的生成。携带 TSG 突变的小 eccDNA 可能作为支持多重 CRISPR/Cas9 诱导的小鼠肝癌肿瘤内异质性和肿瘤进化的重要来源。© 2023。 BioMed Central有限公司，施普林格自然的一部分。

Primary 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.CRISPR/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.After 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.Small 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.© 2023. BioMed Central Ltd., part of Springer Nature.