2025/12/10 by Shunya Hirata, Taisei Ozono, Kenshin Kawai +5 · 1 voice
Agricultural and Biological Sciences · Biochemistry, Genetics and Molecular Biology · Energy · #CRISPR and Genetic Engineering #Metalloenzymes and iron-sulfur proteins #Plant Molecular Biology Research
paper · doi:10.1093/pcp/pcaf162
openalex publication_date 2025/12/10 · openalex created_date 2025/12/11 · openalex updated_date 2026/07/13
DNA methylation is an important epigenetic modification that regulates gene expression and supports genome stability. DNA methylation editing technology differs from conventional genome editing technology, which introduces mutations into genes, in that it enables changing gene expression without altering the base sequence. In this study, we attempted simple and locus-restricted DNA methylation editing in Arabidopsis thaliana using fusion proteins directly linking a nickase-type SpCas9 protein with DNA methylation-related enzymes. First, fusion of the human Ten-eleven translocation methyl cytosine dioxygenase 1 (TET1) catalytic domain (TET1cd) to nSpCas9 led to removing 5-methylcytosine in the FLOWERING LOCUS WA (FWA) promoter region of the wild-type plant, resulting in increased expression of the FWA gene and consequently, a late-flowering phenotype. Conversely, fusion of a mutant form of the bacterial DNA methyltransferase MQ1 (MQ1v) to nSpCas9 induced de novo DNA methylation in the fwa101-D mutant, in which the FWA promoter region is hypomethylated, and suppressed FWA gene expression, resulting in an early-flowering phenotype compared with the fwa101-D mutant. Of particular importance, our nSpCas9 system achieves targeted DNA methylation editing within a genomic window of ~10-20 kb. The nSpCas9 system features a compact and simplified vector structure due to the DNA methylation-related enzyme directly fusing to nSpCas9. Furthermore, sgRNA can be easily replaced, making it highly flexible. We propose a new method for targeted epigenome editing technology in plants, paving the way for innovative strategies in both basic research on epigenetics and crop development through epigenome editing.