BREEDIT: a novel breeding strategy using multiplex genome editing in maize

BREEDIT is a pipeline that combines multiplex genome editing of growth-related genes and gene families with specific crossing schemes.We targeted 48 growth-related genes (GRG) using multiplex CRISPR/Cas9. The 48 GRG were distributed in 4 groups of 12 genes named SCRIPTs, from SCRIPT1 (S1) to SCRIPT4 (S4). Calli from a Cas9-expressing transgenic line named EDITOR were super-transformed using the SCRIPT contructs, each containing 12 gRNAs that target one of the 12 genes of the SCRIPT. The SCRIPT construct and the Cas9 locus were present throughout the generations (from T0 to T2) thus enabling to increase the number of edits. We generated 1740 gene-edited maize seedlings from T0 (calli regeneration: T) to T1 and T2 (selfings: S, backcross to the EDITOR line or maize B104: B, inter- and intra-script crosses: C) that we studied for editing patterns and/or phenotypic traits under well-watered and water-deficient conditions. Maize seedlings were routinely genotyped at the 48 targeted loci and extra putative off-target sites using Illumina amplicon sequencing.