Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut

Abstract

SummaryGenome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.

Using CRISPR/Cas in three dimensions: towards synthetic plant genomes, transcriptomes and epigenomes

“…The simultaneous induction of multiple DSBs opens new avenues for genome engineering. By inducing four DSBs, targeted gene exchange has been induced in plants (Fauser et al ., ; Weinthal et al ., ; Schneider et al ., ). The CRISPR/Cas system is mainly applied to delete plant genomic sequences, e.g., (Zhou et al ., ; Johnson et al ., ) or to simultaneously induce mutations in several genes at the same time, e.g., (Ma et al ., ).…”

Targeted insertion of large DNA sequences by homology‐directed repair or non‐homologous end joining in engineered tobacco BY‐2 cells using designed zinc finger nucleases

“…Electroporation of BY‐2 protoplasts was done as described before (Schneider et al., ). The ratio between donor DNA and ZFN coding DNA was 4:1; the ratio of ZFN2:ZFN4 was 1:1, 1:2, or 1:4.…”

“…The viable cell population was gated based on light scatter signals (SSC‐A and FSC‐H) while the percentage of green fluorescent protoplasts in each culture was detected at 527/32 nm (FITC‐H; GFP). Two types of protoplasts were used to set the gates for the presence of green fluorescence: wild‐type BY‐2 protoplasts and protoplasts derived from TurboGFP expressing cell line C#86 (Schneider et al., ). We analyzed 10 4 viable gated protoplasts for each transgenic culture and processed the signal data with the FACSuite Software (BD Bioscience).…”

“…Cells were kept at room temperature for 2 days to regenerate and distributed to four plates with selective MS medium (100 mg/L kanamycin) for callus formation at 28°C. Electroporation of BY-2 protoplasts was done as described before (Schneider et al, 2016). The ratio between donor DNA and ZFN coding DNA was 4:1; the ratio of ZFN2:ZFN4 was 1:1, 1:2, or 1:4.…”

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].…”