ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation

Abstract

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10−3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10−3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

“…Since the PPO gene is an essential gene, this resulted in stunted growth of the seedlings (Figure S1). This phenotype was not observed in earlier targeted mutagenesis experiments of PPO using ZFNs (de Pater et al 2013), indicating a higher activity of CRISPR/Cas9 on the PPO gene compared to the ZFNs. We noticed that the sgRNA recognized a sequence in the PPO gene with GG just 5′ of the PAM sequence, which was recently shown to promote higher rates of mutagenesis (Farboud and Meyer 2015).…”

Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9

“…2f). Exploration of HDR in Arabidopsis protoplasts was unsuccessful, presumably due to intrinsically low efficiency of HDR in Arabidopsis 18 .…”

Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook

“…The use of ZFNs has certain limitations: the constructs are not easy to design and transform, even in plants, and it is an expensive approach. Moreover, some researchers have reported non-specific nucleotide recognition because of their origin from eukaryotic transcription motifs, making this approach less reliable for genome editing [93,94]. Most restriction nucleases are derived from bacteria and TALENs were isolated from the prokaryotic plant pathogen Xanthomonas [95].…”