RESUMO
Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.
Assuntos
Leucemia Mieloide Aguda , Translocação Genética , Animais , Camundongos , Proteína 1 Parceira de Translocação de RUNX1/genética , Íntrons/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Carga Tumoral , Sistemas CRISPR-Cas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Proliferação de Células , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
CRISPR/Cas9 is a powerful genetic engineering technology that enables the introduction of genomic changes such as deletions and insertions of specific bits of DNA in cells with high precision. Compared to other programmable DNA nuclease such as ZFNs and TALENs, the specific binding of the Cas9 nuclease is mediated by a small guide RNA (gRNA), which can easily be designed to target any locus in the genome. The ease of generating novel gRNA vectors and its high efficiency has rapidly made CRISPR-Cas9 the dominant tool in gene editing applications, including gene knockout, knockin, tagging, etc. Here we describe our method for rapid and efficient generation of gene knockout or deletion cells using CRISPR/Cas9 within the time span of one month. The design of gRNAs, plasmid cloning, transfection, cell culturing, positive clone selection, and screening can be obtained from this method.
