Maize Genomics and Genetics 2024, Vol.15, No.3, 111-122 http://cropscipublisher.com/index.php/mgg 114 GWAS, transcriptomics, CRISPR/Cas9, and epigenomics, researchers can enhance the precision and efficiency of breeding programs, ultimately contributing to increased crop productivity and resilience. Figure 1 The multiplex gene editing strategy of BREEDIT (Adopted from Lorenzo et al., 2022) Image caption: A: Selection of growth-related genes (GRGs) is based on published and in-house research in Arabidopsis, rice, or maize; B: Guide RNAs (gRNAs) with NGG protospacer adjacent motif (PAM) sites are chosen for each gene, PCR primer pairs are designed to resequence gRNA target sites and flanking regions using HiPlex amplicon sequencing; The best sets of gRNAs and primer pairs are selected, and twelve gRNAs are cloned into multiplex gene editing vectors (SCRIPTs); These SCRIPT constructs are then transformed into a Cas9-expressing maize line (EDITOR); C: Vigorous T0 plants containing both the SCRIPT (BASTA resistant) and the Cas9 EDITOR construct (hygromycin resistant) are genotyped using HiPlex amplicon sequencing. Based on the genotypes, plants are selected for various crosses: backcrosses with B104 (BC), intra-script crosses with plants having complementary mutations, and inter-script crosses with plants having different SCRIPTs and mutations in different gene families or pathways; These crosses aim to maximize mutation diversity; Self-crosses (S) produce segregating progeny for high-throughput phenotyping of selected traits, which are then associated with gene combinations; D: Sequencing reads are mapped to the B104 reference loci, deriving two read categories: haplotypeREF (aggregated reads with only SNPs, in-frame indels, or reference haplotype) and haplotypeKO (aggregated reads with out-of-frame indels). HaplotypeKO is divided into three loss-of-function (LOF) genotypic classes based on their tri-modal distribution: LOF0/2 (no out-of-frame indels, 0 chromosomes edited), LOF1/2 (one chromosome edited), and LOF2/2 (both chromosomes edited). (Adapted from Lorenzo et al., 2022)
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