The recent emergence of the CRISPR/Cas system has boosted the possibilities for precise genome engineering approaches throughout all kingdoms of life. The most common application for plants is targeted mutagenesis, whereby a Cas9-mediated DNA double-strand break (DSB) is repaired by mutagenic nonhomologous end joining (NHEJ). However, the site-specific alteration of a genomic sequence or integration of a transgene relies on the precise repair by homologous recombination (HR) using a suitable donor sequence: this poses a particular challenge in plants, as NHEJ is the preferred repair mechanism for DSBs in somatic tissue. Here, we describe our recently developed in planta gene targeting (ipGT) system, which works via the induction of DSBs by Cas9 to activate the target and the targeting vector at the same time, making it independent of high transformation efficiencies.
Keywords: Cas9; Double-strand break repair; Engineered nucleases; Gene technology; Genome engineering.