Gene Replacement by Intron Targeting with CRISPR-Cas9.

The CRISPR-Cas9 system has become the most widely adopted genome editing platform and is used in an expanding number of organisms, mainly by creating targeted knockouts through non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). It would also be highly desirable to be able to use homology-directed repair (HDR) to perform precise gene editing, for example, by replacing a small section of DNA to substitute one amino acid for another in a given gene product. However, this remains a serious challenge in plants. Here, we describe a recently developed intron-mediated site-specific gene replacement method acting through the NHEJ pathway in which Cas9 simultaneously introduces DSBs in adjacent introns and the donor template. This approach is of general use for replacing targeted gene fragments at specific genomic sites in plants.