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Designer nuclease‐mediated gene correction via homology‐directed repair in an in vitro model of canine hemophilia B
Author(s) -
Bergmann Thorsten,
Ehrke-Schulz Eric,
Gao Jian,
Schiwon Maren,
Schildgen Verena,
David Stephan,
Schildgen Oliver,
Ehrhardt Anja
Publication year - 2018
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.3020
Subject(s) - cas9 , nuclease , crispr , gene , homology directed repair , biology , genome editing , microbiology and biotechnology , dna , gene targeting , computational biology , point mutation , plasmid , mutant , genetics , dna repair , dna mismatch repair
Background Gene correction at specific target loci provides a powerful strategy for overcoming genetic diseases. In the present study, we aimed to use an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX ( cFIX ) gene. To correct the defective gene via homology‐directed repair (HDR), we designed transcription‐activator like effector nucleases and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double‐strand breaks at the mutation site. Methods To generate a stable cell line containing the mutated cFIX locus, a 2‐kb genomic DNA fragment derived from a hemophilia B dog was amplified and integrated utilizing the phiC31 integrase system. Designer nucleases were assembled and cloned into vectors for constitutive and inducible expression. To detect mutations, insertions and deletions, and HDR events after nuclease treatment T7E1 assays, an amplification‐refractory mutation system‐quantitative polymerase chain reaction and pyrosequencing were performed. Results To perform HDR correction experiments, we established a cell line carrying the mutated cFIX locus. In HDR approaches we either explored a wild‐type or an optimized cFIX sequence and we found that our modified HDR cassette showed higher gene correction efficiencies of up to 6.4%. Furthermore, we compared inducible and constitutive designer nuclease expression systems and found that the inducible system resulted in comparable HDR efficiencies. Conclusions In conclusion, the present study demonstrates the potential of this strategy for gene therapeutic approaches in vitro and in a canine model for hemophilia B.