| Gene fusion or chromosomal translocation were frequently occurred in human cancers.MLL-AF9 translocation were observed in a number of Acute myeloid leukemia(AML)patients.Construct the cell model of MLL-AF9 is very important for the treatment of AML.CRISPR-Cas9 gene editing technology provides a new tool for precise manipulation of genome.Cmpared with previous gene-editing tools including ZFN or TALEN,CRISPR-Cas9 technology is much more simple and convenient,and it could be used for multiple gene targeting.Several previous studies showed that CRISPR-Cas9 could be used to construct various fusion gene cell models,however the targeting efficiency is lower than 10%,making it very difficult to precisely generate chromosomal translocation.Here we proposed a new strategy to efficiently generate MLL-AF9 fusion gene in vitro by using the CRISPR-Cas9 technology.1.We designed and chose the effective sgRNA in the 10 th intron of MLL gene and the 5th intron of AF9 gene.2.We designed a new targeting donor vector p EASY-Blunt-MLL-GFP-AF9(2Loxp),which contains two homologous arms of MLL and AF9 gene and the sequence of GFP gene.3.Two sgRNAs and targeting vectors were transfected the into HEK293 T cells simultaneously.The homologous recombination were induced between targeting vector and the 10 th intron of MLL gene or the 5th intron of AF9.Finally,the GFP reporter was removed by Cre-Loxp system.4.MLL-AF9 cell clones were identified by long-range PCR and DNA sequencing,and the efficiency was as high as 30%,which greatly improved than previous methods.5.Finally,we tried to confirm the AF9 and MLL gene fusion in HEK293 T cells by fluorescence in situ hybridization technique,but failed,probably due to the cells type we used.Further experiments would be performed in a variety of human cell lines.In summary,we generated a novel gene fusion cell model by using a new strategy.Compared with previous reports,our new strategy are much more efficientand may provide a new approach to genenrate precise chromosomal translocation and gene-fusion in human cells.We anticipated that more genes fusion cell models would be genenrated by using this new strategy in the future. |
