True gene-targeting events by CRISPR/Cas-induced DSB repair of the PPO locus with an ectopically integrated repair template

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.