Related Experiment Video
Updated: Apr 30, 2026
06:26
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
Published on: October 5, 2012
12.4K
Using ngTALEN to improve genome editing efficiency on targets containing 5-methylcytosines
Zhicai Wang1,2, Yoshiko Tamura1, Masaru Hashimoto1
1Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi Bunkyo-ku, Tokyo, 113-8657, Japan.
The Plant Journal : for Cell and Molecular Biology
|April 4, 2026
Summary
DNA methylation impacts genome editing efficiency in plants. Methylated cytosine impedes CRISPR/Cas9 and TALEN editing, but ngTALEN shows promise for targeting methylated DNA sequences.
Area of Science:
- Plant molecular biology
- Epigenetics
- Genome editing technologies
Background:
- Distinct DNA methylation patterns exist between mitochondrial and nuclear-encoded mitochondrial DNA sequences (NUMTs).
- Genome editing efficiency differs between mitochondrial DNA and NUMTs, potentially due to methylation status.
- Transcription activator-like effector nucleases (TALEN) offer efficient genome editing, with modified versions targeting specific DNA modifications.
Purpose of the Study:
- To investigate the hypothesis that DNA methylation status affects genome editing outcomes.
- To compare the editing efficiency of CRISPR/Cas9, TALEN, and a novel ngTALEN system in Arabidopsis thaliana.
- To assess the impact of 5-methylcytosine (5mC) on editing at methylated and unmethylated loci.
Main Methods:
- Utilized ngTALEN, engineered to recognize 5-methylcytosine (5mC), to target the Flowering Wageningen (FWA) locus in Arabidopsis thaliana.
- Targeted promoter and gene body regions with varying levels of cytosine methylation.
- Performed comparative analysis using CRISPR/Cas9 and TALEN systems across different methylation states and genomic loci.
Main Results:
- 5mC impeded editing by both CRISPR/Cas9 and TALEN at the FWA promoter and gene body.
- TALEN demonstrated robust editing comparable to CRISPR/Cas9 across multiple sites.
- ngTALEN exhibited improved editing efficiency at CG-hypermethylated FWA promoter compared to TALEN, but showed reduced effectiveness at loci with differing methylation and chromatin states.
Conclusions:
- DNA methylation significantly hinders genome editing in plants, affecting both CRISPR/Cas9 and TALEN systems.
- ngTALEN represents a specific and robust tool for enhancing genome editing at CG-methylated sites.
- This study provides the first systematic comparison of CRISPR/Cas9 and TALEN editing efficiency on methylated DNA in plants.
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