Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Abstract

Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated sitespecific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFNmediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

Making ends meet: targeted integration of DNA fragments by genome editing

“…One route that has been implemented to improve efficiency is to couple NHEJ with ligation in vivo (“end-capture”) of compatible overhangs created in the donor DNA and the genomic target site by molecular scissors such as ZFNs (Fig. 3) [Orlando et al 2010; Cristea et al 2013; Weinthal et al 2013]. The cohesive ends of the donor and target can pair, resulting in a product with the donor inserted at a single orientation.…”

Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

“…By fusing the DNA cleavage domain from the restriction enzyme Fok I to the highly variable DNA binding domain (DBD) of different zinc finger transcription factors to form different ZFNs, different target sites in the DNA can be recognized and cleaved ( Figure 1A ) (Kim et al, 1996). GT was demonstrated to be feasible in Arabidopsis and tobacco by inducing a DSB with a ZFN (Wright et al, 2005; Cai et al, 2009; de Pater et al, 2009, 2013; Townsend et al, 2009; Even-Faitelson et al, 2011; Qi et al, 2013; Weinthal et al, 2013; Baltes et al, 2014). So far, only two cases reported the successful GT for integration or stacking herbicide resistances gene(s) in a crop plant (maize).…”

Plant Genome Editing and its Applications in Cereals

“…The NHEJ pathway is usually recognized with many errors and, therefore, is an excellent choice for gene disruption. NHEJ can achieve all editing objectives, i.e., mutations including small deletions or insertions [22], as well as gene insertion and gene replacement [19,23]. However, while geting a mutation is certain, the mutations are completely random, and unlike the homologous directed recombination (HDR), there is no way to predict which mutation will occur and what will be the inal result.…”

“…Therefore, one can use oligonucleotides such as triplex forming oligonucleotides (TFOs), short ssDNA or dsDNA donors such as T-DNA to induce HR. It should be noted that while T-DNA of Agrobacterium is iniltrated in the plant cell as a ssDNA coated by VirE2, it turns into dsDNA shortly after and probably integrates into the plant genome as dsDNA [19,20]. While unassisted HR levels are very low, and it is highly induced by speciic genomic DSBs [21].…”