Using <scp>ngTALEN</scp> to improve genome editing efficiency on targets containing 5‐methylcytosines

We recently discovered distinct methylation patterns between the mitochondrial genome and the nuclear-encoded mitochondrial DNA sequences (NUMTs), with the mitochondrial genome being hypomethylated and NUMTs being hypermethylated. Given that genome editing using mitochondrial targeted transcription activator-like effector nucleases (TALEN) is highly efficient, while editing at NUMT is difficult, we hypothesized that the methylation status might affect editing outcomes. To test this, we attempted to use ngTALEN [employing RVD-NG to recognize 5-methylcytosine (5mC)] to target the Flowering Wageningen (FWA) locus of Arabidopsis thaliana, specifically the promoter and gene body regions with varying levels of cytosine methylation. Comparative analysis using the active epimutant allele fwa-d and wild-type Columbia-0 (Col-0) carrying a naturally silenced allele of FWA revealed that editing was impeded by 5mC at both the promoter and gene body of FWA for both CRISPR/Cas9 and TALEN. Overall, TALEN editing is robust and comparable to that of CRISPR/Cas9 at multiple sites, while ngTALEN showed improved editing at the CG-hypermethylated promoter of FWA compared with TALEN. Additionally, when targeting multiple genomic loci with identical sequences that differ in methylation levels and chromatin states, ngTALEN was less effective to induce edits. Therefore, this study represents the first systematic comparison of editing efficiency between CRISPR/Cas9 and TALEN in dealing with methylated or unmethylated DNA in plants. Furthermore, we have developed ngTALEN as a specific and robust tool for enhancing editing at sites with various levels of CG methylation.