Abstract
In recent years, several tools have become available for improved gene-targeting (GT) in plants. DNA breaks at specific sites activate local DNA repair and recombination, including recombination with ectopic sequences leading to GT. Large-scale transformation with the repair template can be avoided by pre-insertion of the repair template in the genome and liberation by sequence-specific nucleases (in planta GT procedure). Here, we tested whether release of the repair template was required for GT. Plants were transformed with constructs encoding a CRISPR/Cas nuclease with a recognition site in the endogenous PPO gene and a repair template harboring a 5′ truncated PPO gene with two amino acid substitutions rendering the enzyme insensitive to the herbicide butafenacil. Selection resulted in so-called true GT events, repaired via homologous recombination at both ends of the gene and transmitted to the next generation. As the template was surrounded by geminiviral LIR sequences, we also tested whether replication of the template could be induced by crossing-in an integrated geminivirus REP gene. However, we could not find evidence for repair template replication by REP and we obtained similar numbers of GT events in these plants. Thus, GT is possible without any further processing of the pre-inserted repair template.
Highlights
One of the tools that is of great value for fundamental and applied research is the precise modification of DNA sequences
Our strategy for in planta GT employed a pre-inserted CRISPR/Cas nuclease gene for double strand breaks (DSBs) induction at the genomic target site and a pre-inserted repair template surrounded by bean yellow dwarf virus (BeYDV) geminivirus large and short intergenic regions (LIR and SIR)
This has been achieved by transformation with repair templates surrounded by geminivirus LIR sequences on which the viral REP protein acts leading to multicopy amplification by rolling circle replication[26]
Summary
One of the tools that is of great value for fundamental and applied research is the precise modification of DNA sequences. As only a limited number of cells will be transformed, in a newer procedure, called in planta GT, the repair template was pre-inserted (as T-DNA) in the genome and released in plants containing the construct in all of its cells where sequence-specific nucleases www.nature.com/scientificreports/. The geminivirus bean yellow dwarf virus (BeYDV) was used for delivery of nuclease genes and repair templates in tobacco, tomato and wheat[26,27,28] In this way, replication of the repair template was observed to enhance the GT frequency[26]. We showed that DSB-induction in the natural PPO gene using ZFNs enhanced GT5 In these experiments, homologous recombination between the endogenous PPO gene and the repair construct occurred directly after delivery of the repair template as T-DNA by Agrobacterium floral dip transformation.
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