Extrachromosomal circular DNA harboring targeted tumor suppressor gene mutations supports intratumor heterogeneity in mouse liver cancer induced by multiplexed CRISPR/Cas9

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. Here, using this approach targeting 34 tumor suppressor genes (TSGs), we efficiently induced 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 intratumor genetic heterogeneity (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 extrachromosomal circular DNA (eccDNA) harboring targeted TSG mutations in these tumor cells. These findings suggest an important contribution of eccDNA carrying TSG mutations to tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.