NEW YORK (GenomeWeb) – Using two relatively new technologies — CRISPR-Cas9 genome editing and organoid culture techniques — researchers in the Netherlands and the UK have identified a specific DNA repair defect as the underlying cause of a mutational signature found in certain cancers.
As they reported today in Science, the researchers used CRISPR-Cas9 to delete key DNA repair genes in human colon organoids, which are small stem cell-derived cultures that mimic many features of intact intestinal tissue.
"Organoid technology allows for the long-term in vitro expansion of epithelial tissues, starting from a single adult stem cell. Such organoids remain genetically stable over long periods of time. We have previously shown that clonal organoid cultures can be used for in-depth pattern analysis of mutations that accumulate throughout life in tissue-specific adult stem cells," the authors wrote.
The editing of the organoids was followed by delayed subcloning and whole-genome sequencing, after which the researchers found that mutations had accumulated in the organoids deficient in the mismatch repair gene MLH1. The mutations were driven by replication errors and their accumulation accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers, the authors wrote.
They then applied this strategy of CRISPR genome editing in colon organoids to the cancer predisposition gene NTHL1, which encodes a base excision repair protein. This experiment revealed a mutational footprint designated signature 30, which had previously been observed in breast cancer, but which had been unexplained up to now.
"Signature 30 has previously been identified in one patient with breast cancer interrogated by [whole-genome sequencing]," the researchers wrote. "We examined tumor and germline sequences of this breast cancer patient and identified a germline nonsense mutation in NTHL1, compounded by loss of heterozygosity in the tumor. This observation further corroborates the link between NTHL1 deficiency and signature 30."
The findings show that mutational signatures of certain cancers can be ascertained through genomic characterization of CRISPR-edited human organoid subclones, the team added. The engineered MLH1 organoids validated the approach, while the subsequent experiment showed that a high contribution of signature 30 mutations within a tumor can indicate cancer-predisposing germline mutations in NTHL1, and that the signature may indicate predisposition to a broader range of cancers than previously thought.
"Indeed, NTHL1 germline mutations predispose not only to breast cancer, but to other cancer types including colorectal cancer," the researchers wrote. "The strategy we have described can be used to study the mutational consequences of DNA repair knock-outs or mutagen exposure, to systematically dissect mutational signatures and potentially unveil their molecular origins."