Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.
Keywords: AML; CRISPR-directed mutagenesis; FLT-3.