Barriga et al. develop MACHETE, a genome engineering strategy enabling the flexible modeling of megabase-sized deletions and show that the concomitant loss of the interferon cluster with CDKN2A/B deletions on 9p21.3 enhances immune evasion.
of penicillin/streptomycin , 100 mM Glutamax , ITS supplement , 0.1 mg mlcholera toxin , 4 μg mlTumor-derived cell lines were generated by an initial mechanical disaggregation/mincing and tumor fragments were transferred to a solution of type V collagenase and incubated at 37 °C for 45 min. Cell suspensions were supplemented with an equal volume of DMEM 10% FBS and filtered through a 100-μm mesh . Filtered suspensions were centrifuged for 5 min at 300and cultured on collagen-coated plates .
5 μg of donor DNA was added to the Cas9 RNP complex, which was then mixed with the cell aliquot. The cell–RNP–donor mixture was electroporated . For the selection of cassette KI lines, puromycin was added to the medium 48 h after electroporation. In the case of fluorescence reporters, cells were sorted 48 h after electroporation and further enriched for stable expression 1 week after this initial sort. Selected cells were expanded to establish the parental KI lines.
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