TALEN mediated gene editing in a mouse model of Fanconi anemia

Maria José Pino-Barrio , Yari Giménez, Mariela Villanueva , Marcus Hildenbeutel , Rebeca Sánchez-Dominguez , Sandra Rodríguez-Perales , Roser Pujol , Jordi Surrallés , Paula Río  , Toni Cathomen , Claudio Mussolino , Juan Antonio Bueren , Susana Navarro
Source: Scientific Reports
Publication Date: (2020)
Issue: 10(1): 6997
Research Area:
Gene Expression
Regenerative medicine
Cells used in publication:
Bone marrow, mouse
Species: mouse
Tissue Origin: bone marrow
Hematopoietic stem cells, mouse
Species: mouse
Tissue Origin: bone marrow
Embryonic fibroblast, mouse (MEF)primary
Species: mouse
Tissue Origin: embryo
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit

For nucleofection of WT or FA-A immortalized MEFs, 2 × 106 cells per condition were used with the Amaxa MEF2 Nucleofector® Kit (Lonza Group) using program T20 of the Nucleofector™ I device, 2.5 µg of each TALE N monomer were nucleofected together with a determined donor dose (0.75, 2 and 4 µg). Enrichment during 5 passages with puromycin (1–1.25 µg/ml) was performed, then clones were generated by limiting dilution to perform gene targeting studies. For the BM hematopoietic cells, 1.4 × 106 cells of purified BM Lin- cells were nucleofected with an EGFP control plasmid or the corresponding doses of the TALEN monomers and donors after pre-stimulation during 48 h. Lin- BM cells were nucleofected with the 4-D Nucleofector™ device using the P3 Primary Cell 4D-Nucleofector® X Kit (Lonza Group) kit with the ED-113 program.


The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.