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Tytuł pozycji:

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Tytuł :
Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining
Autorzy :
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.
Pokaż więcej
Temat :
soybean
food and beverages
genome editing
Research Article
fungi
somatic embryogenesis
gene targeting
zinc finger nuclease
biolistic transformation
Research Articles
Źródło :
Plant Biotechnology Journal
Wydawca :
John Wiley and Sons Inc., 2018.
Rok publikacji :
2018
Oryginalny identyfikator :
pmc: PMC6419576
pmid: 30220095
Język :
English
ISSN :
1467-7652
1467-7644
DOI :
10.1111/pbi.13012
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 (DSBs) at user‐specified genomic loci. These DSBs 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 DSBs 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 (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 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 ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.
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