Open AccessZinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining
Author(s) -
Bonawitz Nicholas D.,
Ainley W. Michael,
Itaya Asuka,
Chennareddy Sivarama R.,
Cicak Tobias,
Effinger Katherine,
Jiang Ke,
Mall Tejinder Kumar,
Marri Pradeep Reddy,
Samuel J. Pon,
Sardesai Nagesh,
Simpson Matthew,
Folkerts Otto,
Sarria Rodrigo,
Webb Steven R.,
Gonzalez Delkin O.,
Simmonds Daina H.,
Pareddy Dayakar R.
Publication year - 2019
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.13012
Subject(s) - zinc finger nuclease , genome editing , biology , crispr , genetics , gene targeting , non homologous end joining , cas9 , transgene , homology directed repair , zinc finger , locus (genetics) , nuclease , gene , genome engineering , transcription activator like effector nuclease , homologous recombination , computational biology , dna repair , transcription factor , dna mismatch repair
Summary Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks ( DSB s) at user‐specified genomic loci. These DSB s subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean ( Glycine max ) via non‐homologous end joining ( NHEJ )‐mediated repair of such DSB s has been previously demonstrated with multiple nucleases, as has homology‐directed repair ( HDR )‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR /Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease ( ZFN ). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a ( FAD 2‐1a ) locus of embryogenic cells in tissue culture. We then describe ZFN ‐ and NHEJ ‐mediated, targeted integration of two different multigene donors to the FAD 2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T 1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFN s can be used to generate fertile transgenic soybean plants with NHEJ ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.