CRISPR-Cas9 is now a common tool for targeted mutagenesis and genomic knockouts in numerous crops. However, its efficiency for gene targeting (GT) via homologous recombination (HR) is still low. HR-mediated GT requires the generation of DNA double-strand break (DSB) in the genomic target site synchronized with the supply of a donor DNA repair template containing flanking regions of homology. Here, we report an intra-genomic approach in maize using a heat-shock inducible Cas9 expression that results in high-frequency, selectable marker-free GT. The T-DNA vector used contained a single guide RNA, an inducible Cas9 expression cassette, a donor DNA repair template flanked by Cas9 cut sites matching the genomic target site, and morphogenic genes (<i>Bbm</i> and <i>Wus</i>). In addition, the donor DNA repair template is placed between a promoter and a selectable marker (SM) coding sequence, rendering the SM non-functional. The first step of the process is the generation of the donor locus by random integration of the T-DNA in the genome. Induction by heat-shock of Cas9 expression during early stages of maize transformation results in 1) DSB at the genomic target site, 2) the mobilization of the repair template intra-genomically, and 3) activation of the SM upon donor template excision within the donor locus.  The activation of the selectable marker gene provides a preferential selection for plant regeneration only from cells with released donor template and potential GT. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants. Analysis of T1 progeny confirmed Mendelian inheritance and segregation of the GT-modified target locus

Intra-genomic gene targeting in maize using inducible CRISPR-Cas9

ACS Fall 2021

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