Synthetic nucleases for genome engineering in plants: prospects for a bright future

Abstract

By inducing double-strand breaks (DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a means to target DSBs to specific sites. This limitation was overcome by development of modified meganucleases and synthetic DNA-binding domains. Domains derived from zinc-finger transcription factors or transcription activator-like effectors may be designed to recognize almost any DNA sequence. By fusing these domains to the endonuclease domains of a class II restriction enzyme, an active endonuclease dimer may be formed that introduces a site-specific DSB. Recent studies demonstrate that gene knockouts via non-homologous end joining or gene modification via homologous recombination are becoming routine in many plant species. By creating a single genomic DSB, complete knockout of a gene, sequence-specific integration of foreign DNA or subtle modification of individual amino acids in a specific protein domain may be achieved. The induction of two or more DSBs allows complex genomic rearrangements such as deletions, inversions or the exchange of chromosome arms. The potential for controlled genome engineering in plants is tremendous. The recently discovered RNA-based CRISPR/Cas system, a new tool to induce multiple DSBs, and sophisticated technical applications, such as the in planta gene targeting system, are further steps in this development. At present, the focus remains on engineering of single genes; in the future, engineering of whole genomes will become an option.

Keywords: double-strand break repair; gene targeting; gene technology; synthetic nucleases; targeted mutagenesis.