Lakshmi Sastry-Dent

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

Genome 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.

Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.

Identification and use of the sugarcane bacilliform virus enhancer in transgenic maize

BackgroundTranscriptional enhancers are able to increase transcription from heterologous promoters when placed upstream, downstream and in either orientation, relative to the promoter. Transcriptional enhancers have been used to enhance expression of specific promoters in transgenic plants and in activation tagging studies to help elucidate gene function.ResultsA transcriptional enhancer from the Sugarcane Bacilliform Virus - Ireng Maleng isolate (SCBV-IM) that can cause increased transcription when integrated into the the genome near maize genes has been identified. In transgenic maize, the SCBV-IM promoter was shown to be comparable in strength to the maize ubiquitin 1 promoter in young leaf and root tissues. The promoter was dissected to identify sequences that confer high activity in transient assays. Enhancer sequences were identified and shown to increase the activity of a heterologous truncated promoter. These enhancer sequences were shown to be more active when arrayed in 4 copy arrays than in 1 or 2 copy arrays. When the enhancer array was transformed into maize plants it caused an increase in accumulation of transcripts of genes near the site of integration in the genome.ConclusionsThe SCBV-IM enhancer can activate transcription upstream or downstream of genes and in either orientation. It may be a useful tool to activate enhance from specific promoters or in activation tagging.

Zinc Finger Nuclease-Mediated Gene Targeting in Plants

The ability to generate DNA double-strand breaks (DSBs) at specified locations in a plant’s genome, thereby stimulating the cell’s DNA repair processes, represents a promising means of facilitating genetic modification for both basic studies of gene function as well as applied crop improvement. Zinc finger nucleases (ZFNs) are engineered restriction enzymes consisting of a nonspecific cleavage domain and sequence-specific DNA-binding domains designed to create site-specific DSBs. Since DSB repair in plants appears to occur primarily via error-prone nonhomologous end joining (NHEJ) processes, ZFNs designed to cleave endogenous genes is a path toward targeted mutagenesis. Similarly, ZFN-mediated induction of concurrent DSBs can give rise to targeted deletions of genomic segments between cleavage sites. In addition, homology-directed repair of targeted DSBs allows for site-specific transgene integration into transgenic and endogenous gene loci as well as the creation of specific sequence modifications. The combination of sequence-specific DNA cleavage by designed ZFNs and homology-directed DSB repair at investigator-specified break sites makes precision genome modification a reality. This capability, in combination with rapid advances in genome sequencing and bioinformatics, bodes well for the future of plant functional genomics and crop improvement.

Use of Zinc-Finger Nucleases for Crop Improvement

Over the past two decades, new technologies enabling targeted modification of plant genomes have been developed. Among these are zinc-finger nucleases (ZFNs) which are composed of engineered zinc-finger DNA-binding domains fused with a nuclease, generally the FokI nuclease. The zinc-finger domains are composed of a series of four to six 30 amino acid domains that can bind to trinucleotide sequences giving the entire DNA-binding domain specificity to 12-18 nucleotides. Since the FokI nuclease functions as a dimer, pairs of zinc-finger domains are designed to bind upstream and downstream of the cut site which increases the specificity of the complete ZFN to 24-36 nucleotides. The ability of these engineered nucleases to create targeted double-stranded breaks at designated locations throughout the genome has enabled precise deletion, addition, and editing of genes. These techniques are being used to create new genetic variation by deleting or editing endogenous gene sequences and enhancing the efficiency of transgenic product development through targeted insertion of transgenes to specific genomic locations and to sequentially add and/or delete transgenes from existing transgenic events.