Figure 4.

The directed evolution approach for synthetic promoter generation in vivo.

Based on the cis elements of native plant promoters, gRNA libraries for gene editing are synthesized and ligated into a CRISPR-Cas9 vector. The positive plasmids are co-transformed into Agrobacterium to create a library with multiple editing sites for use in Agrobacterium infection. Large-scale Agrobacterium-mediated transformation is performed to generate plant populations with directed mutated promoters for trait assessment in the field. To reduce the number of genetic transformations, F₁ plants carrying Cas9 and gRNAs are selected and crossed with the wild type (WT) to generate larger plant populations with direct-mutated promoters. After field testing and trait assessment, the genetically modified plants with evolved promoters are chosen as germplasms for plant biotechnology or breeding. Triangles show the binding sites of gRNAs. +1 indicates the TSS.