Fig. 1: A series of tools target DNA methylation to the FWA promoter.

a Design of dCas9 fusion proteins for editing DNA methylation. Effector and cofactor proteins were fused to the N and C terminus of dCas9, respectively. The pUBQ1 and pRPS5A promoters were chosen to drive the expression of the designed fusion proteins. b Schematic of targeting DNA methylation at the FWA promoter. This methylation-dependent visual phenotype can be used as a readout for successful targeted DNA methylation. c fwa plants and representative early-flowering T1 plants expressing MQ1v-dCas9-KRAB (M22) to illustrate differences in flowering-time. d Bar graph showing FWA expression levels in representative early-flowering T1 plants expressing the indicated tools and control plants. The controls and T1 plants were grown side by side. Error bars are defined as the standard deviation (SD) of three technical replicates; data are presented as mean ± s.d.. e Dot plot of rosette leaf count at flowering-time of T1 plants transformed with indicated tools in fwa, compared to fwa controls. The rosette leaf count reflects the time required for flowering. “-gRNA” indicates the transformed vector without the gRNA expression cassette (the same below). pU-dCas9 indicates that the pUBQ1 promoter drive the expression of single dCas9 protein (the same below); pR-dCas9 indicates that the pRPS5A promoter drive the expression of single dCas9 protein (the same below). Each dot represents the rosette leaf number of an individual plant (the same below). The dashed line corresponds to the leaf number threshold (21) below which plants are considered early-flowering relative to fwa plants. The code names referring to the tools are listed below. The numerator in the fraction at the top indicates the number of early-flowering plants relative to fwa plants, while the denominator represents the total number of plants observed (the same below). The controls and T1 plants were grown side by side. f Screenshot of DNA methylation over FWA promoter in the indicated T1 plants. The grey box highlights the gRNA-targeted site at the FWA promoter (the same below). Red bars indicate the positions of the gRNAs (the same below). The controls and T1 plants were grown side by side. g Genome-wide CG methylation levels in the indicated T1 and fwa plants. Source data are provided as a Source data file.