Publications
Protocol
The elimination of large genomic regions has been enabled by the advent of site-specific nucleases. However, as the intended deletions get larger, the efficiency of successful engineering decreases to a point where it is not feasible to retrieve edited cells due to the rarity of on-target events. To address this issue, we developed a system called molecular alteration of chromosomes with engineered tandem elements (MACHETE). MACHETE is a CRISPR–Cas9-based system involving two stages: the initial insertion of a bicistronic positive/negative selection cassette to the locus of interest. This is followed by the introduction of single guide RNAs flanking the knockin cassette to engineer the intended deletion, where only cells that have lost the locus survive the negative selection. In contrast to other approaches optimizing the activity of sequence specific nucleases, MACHETE selects for the deletion event itself, thus greatly enriching for cells with the engineered alteration. The procedure routinely takes 4–6 weeks from design to selection of polyclonal populations bearing the deletion of interest. We have successfully deployed MACHETE to engineer deletions of up to 45 Mb, as well as the rapid creation of allelic series to map the relevant activities within a locus. This protocol details the design and step-by step procedure to engineer megabase-sized deletions in cells of interest, with potential application for cancer genetics, transcriptional regulation, genome architecture and beyond.
Tools of the Trade
In this brief piece, Pancho highlights the development of MACHETE and the insights on CNA biology enabled by this new approach.
Article
The most prominent homozygous deletions in cancer affect chromosome 9p21.3 and eliminate CDKN2A/B tumor suppressors, disabling a cell-intrinsic barrier to tumorigenesis. Half of 9p21.3 deletions, however, also encompass a type I interferon (IFN) gene cluster; the consequences of this co-deletion remain unexplored. To functionally dissect 9p21.3 and other large genomic deletions, we developed a flexible deletion engineering strategy, MACHETE (molecular alteration of chromosomes with engineered tandem elements). Applying MACHETE to a syngeneic mouse model of pancreatic cancer, we found that co-deletion of the IFN cluster promoted immune evasion, metastasis and immunotherapy resistance. Mechanistically, IFN co deletion disrupted type I IFN signaling in the tumor microenvironment, leading to marked changes in infiltrating immune cells and escape from CD8+ T-cell surveillance, effects largely driven by the poorly understood interferon epsilon. These results reveal a chromosomal deletion that disables both cell-intrinsic and cell -extrinsic tumor suppression and provide a framework for interrogating large deletions in cancer and beyond.