CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis

Abstract

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.

Murine Models of Acute Myeloid Leukemia

“…However, KMT2A-MLLT1 fusions are much more common in ALL and occur rarely in AML, limiting the use of these models in AML research. Finally, CRISPR/Cas9-mediated genome editing has been used to create a model of Kmt2a-Mllt3 AML via dual single-guide RNAs simultaneous targeting the breakpoint cluster region of Kmt2a and Mllt3 ( 67 ). Unique to KMT2A r AML, close attention must be paid to the model system and cell of origin in studying KMT2A r disease, as similar KMT2A fusions can be found across hematologic malignancies, and selection of methods will have profound effects on the resulting disease.…”

Creation of Philadelphia chromosome by CRISPR/Cas9-mediated double cleavages on BCR and ABL1 genes as a model for initial event in leukemogenesis

“…45 Subsequently, EWSR::FLI1 and NPM1::ALK were generated by simultaneous cleavages at the target sites with the CRISPR/Cas9 system. [46][47][48][49] Similarly, leukemogenic MLL fusion genes including MLL::AF4, [50][51][52] MLL::AF9, 50,[53][54][55] and MLL::ENL 56 were generated by double cleavages using CRISPR/Cas9 in human hematopoietic cells and in the murine 32D myeloid progenitor cell line. For the development of incorrect ligation to take place between two DSBs, each DSB is supposed to be proximally located in the nucleus.…”

Viewing AML through a New Lens: Technological Advances in the Study of Epigenetic Regulation

“…Unlike other models of MLL-r leukemia, which often rely upon the exogenous expression of MLL::FP, the generation of endogenous translocations closely recapitulates the events leading to leukemogenesis in patients. Murine in vitro Mll::Af9 models of MLL-r leukemia have also been generated in murine hematopoietic stem and progenitor cells (HSPCs) using this technology [ 71 ]. In addition to human CD34+ cord blood cells, one model of human MLL::AF4 infant ALL was performed by editing fetal HSPC cells [ 69 ].…”