DNA methylation analysis and editing in single mammalian oocytes

Area of Science:

• Epigenetics
• Developmental Biology
• Reproductive Science

Background:

• Mammalian oocytes transmit essential nongenetic information, including DNA methylation, impacting offspring development and disease susceptibility.
• Current methods for analyzing and manipulating oocyte-specific methylation are limited, hindering functional studies and therapeutic applications.

Purpose of the Study:

• To develop and validate methods for evaluating and manipulating DNA methylation in single mammalian oocytes.
• To investigate the utility of first polar body (PB1) methylation profiles for predicting oocyte methylation status.
• To demonstrate the potential of targeted methylation editing for correcting developmental defects and diseases.

Main Methods:

• Optimization of a single-cell bisulfite sequencing technique for high-efficiency DNA methylation analysis.
• Utilizing the first polar body (PB1) as a proxy for oocyte methylation analysis.
• Employing microinjection-mediated dCas9-Tet/Dnmt-based targeted methylation editing in single oocytes.

Main Results:

• PB1 methylation profiles accurately reflect methylation patterns in sibling oocytes at specific genomic regions.
• Single-cell bisulfite sequencing of PB1 enabled efficient evaluation of methylation related to coat color and parthenogenetic development competency.
• Targeted methylation editing successfully reversed coat color phenotypes, enabled bimaternal mouse development, and corrected Angelman syndrome in a mouse model.

Conclusions:

• The developed methods allow for precise evaluation and manipulation of DNA methylation in single oocytes and their PB1s.
• This approach provides a powerful tool for investigating the role of oocyte epigenetics in development and disease.
• The findings offer a promising strategy for preventing and treating maternally transmitted nongenetic disorders.