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.
Highlights
Rearrangements of the mixed lineage leukaemia gene (MLLr) are the most common cytogenetic abnormalities found in infant and childhood acute lymphocytic leukaemia (ALL) and acute myeloid leukaemia (AML) [1] and are found in 80% and 35–50% of all paediatric ALL and AML cases respectively [1]
We utilized a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system for the generation of murine t(4;9) MA9 chromosomal translocations in murine haematopoietic stem and progenitor cells (HSPCs), utilizing simultaneous targeting of the Mll and Af9 genes by CRISPR single guide RNA (sgRNA) directed to chromosomes
By rational design of sgRNA flanking breakpoint regions with common occurrence in MLL rearrangements (MLLr) AML patients and prior MA9 transgenic models, we identified optimal combinations of sgRNA sequences with which to induce simultaneous double strand breaks (DSBs) at the specific loci
Summary
Rearrangements of the mixed lineage leukaemia gene (MLLr) are the most common cytogenetic abnormalities found in infant and childhood acute lymphocytic leukaemia (ALL) and acute myeloid leukaemia (AML) [1] and are found in 80% and 35–50% of all paediatric ALL and AML cases respectively [1]. Up to 135 MLL FPGs have been identified and are well described elsewhere [5,6,7] These include cytoplasmic proteins (AF6, GAS7 and EEN), septins (SEPT2, SEPT5, SEPT6, SEPT9 and SEPT11) and histone acetyltransferases (CBP and P300) as well as the largest subset, the nuclear proteins. This category contains the most common FPGs such as ENL, ELL, AF4, AF9 and AF10, which account for approximately 80% of all MLL fusions [5]. MLL-AF9 (MA9) t(9;11)(p22;q23) translocations are found in 30% of MLLr AML cases [5] irrespective of age and are commonly found in a subset of adult patients with therapy-related AML [8]
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