Targeted Disruption Of FLT3Juxtamembrane Domain Using TALENs:the Mechanisms And Effects

Objective:Hematologic malignancies are often caused by multiple complex chromosomal aberrations and/or gene mutations. Biological effects caused by a single mutation are often veiled by complicated genetic background. The rapid development of genome editing tools enables convenient targeted knock-in or knock-out of predesigned DNA sequences in multiple biological systems within a short period of time. Thus rapid generation of isogenic cellular disease models, with completely controllable genetic background by precise genome modification with TALENs, provides great value to the study of mutation related pathogenesis and the development of new therapeutics.Methods:In this study, appropriate target site and multiple TALENs were designed against human FLT3and SF3B1gene and zebrafish Sema4d gene. TALENs were constructed via modified Golden-gate cloning method and verified by sequencing. Donor DNA template (with or without cre-loxp antibiotic selection casette) was cloned from primary samples or generated by site mutagenesis for homologous recombination of FLT3-ITD mutation or SF3B1K700E point mutation.’Traffic light’dual fluorescence protein reporter and enrichment system were constructed for SF3B1targeting. Plasmids encoding TALENs or DNA template were nucleofected into target cell-line K562. Subcloning of cell population and PCR verification were used to identify genetic FLT3knock-out clones, and antibiotic selection and Cre recombinase treatment were used to separate FLT3-ITD knocked-in clones. SF3B1knock-in clones were enriched by’traffic light’reporter system followed by cell sorting and sequencing verification. In vitro transcribed mRNAs encoding TALENs were micro-injected into one-cell stage zebrafish embryos, and genomic DNA was extracted to evaluate TALENs efficacy by PCR and T vector cloning. Results:The designed TALENs against predetermined targets exhibited robust cleavage effect in living cells with efficiencies varied from3%to26%(unnormalized, by different detection method). Several heritable K562subclones with disrupted FLT3in single allele, two heterozygous FLT3-ITD knock-in subclones, two heterozygous SF3B1K700E subclones and multiple wild type control clones were obtained. Chimera founder zebrafish(FO) model with genetically disrupted sema4d was established.Conclusion:TALENs as a programmable, convenient and robust genome editing tools compatible with versatile biology systems, in combination with donor DNA template and/or ’traffic light’ reporter and enrichment system, was sufficient to generate cell-based isogenic disease model and chimera zebrafish gene knock out model, thus providing excellent platforms for studies of gene mutation related biologic mechanisms and effects and corresponding therapies. Objective:As one of the most frequently mutated genes in AML, FLT3-ITD (internal tandem duplication) or TKD (tyrosine kinas domain) mutation related aberrant downstream signaling activation and the wild type FLT3receptor overexpression exert significant impact on the clinical prognosis of AML. So far, the targeted inhibition methods of FLT3tyrosine activity are restricted to small molecular compounds, neutralizing antibodies and RNA interference. To obtain specific and long term inhibitory effects of FLT3, we try to target FLT3gene in leukemia cells using genome editing tool TA LENs (transcription activator like effector nucleases), and to assess the biological effect caused by targeted knock-out of FLT3.Methods:TALENs were designed against the sequence encoding juxtamembrane domain (JM) and constructed using modified rapid assembly method. K562cells were nucleofected with plasmids encoding TALENs and subcloned to PCR screening for FLT3knock out clones.20most possible off-target genome loci were examined by PCR reaction and sequencing. Primers expanding different exons were used in RT-PCR to examine the FLT3 transcripts level. Immunoprecipitation was performed to assess the phosphorylation of FLT3receptors and the phosphorylation levels of canonical downstream pathway singnaling were assessed by Western blot analyses. CCK8assay, CSFE staining and colony forming assay were applied to test the proliferation and colony forming abilities of K562clones with FLT3haploinsufficiency in vitro. Xenograft leukemia models in NOD/SCID mice were established using one K562clone with disrupted FLT3in single allele and one K562clone with wild wild type FLT3as control to evaluate biological effects of FLT3haploinsufficiency to leukemia cells in vivo.Results:Designed and assembled TALENs targeting FLT JM domain exhibited robust genome cleavage activity and K562clones with frame shift mutations in FLT3gene in single allele were obtained, and no off-target effect was detected by extensive PCR and sequencing results. Haploinsufficiency caused by frame shift mutation in single allele FLT3resulted in decreased phosphorylation of FLT3receptor and downstream signal transducer AKT, ERK and STAT5. The proliferation or colony forming ability was impaired in the K562clones with single intact FLT3gene comparing to the wild type control clone. In NOD/SCID mice xenograft leukemia model, delay of disease onset, improved survival rate and ameliorated phenotype were observed in K562cells with FLT3haploinsufficiency.Conclusion:Specific disruption of genome sequence encoding JM domain of FLT3receptor using TALENs could inhibit FLT3tyrosine kinase activity, possibly due to the haploinsufficiency of FLT3resulting in blockage of FLT3receptor phosphorylation and downstream signal pathways. Gene disruption in frequently mutated FLT3JM domain can inhibited leukemia cell proliferation both in vitro and in vivo, which provides novel insight into the FLT3tyrosine kinase activity targeting strategy.