Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system

Area of Science:

• Molecular Biology
• Epigenetics
• Gene Regulation

Background:

• Establishing causal links between epigenetic modifications and gene transcription necessitates precise molecular tools.
• CRISPR/dCas9 technology offers a versatile platform for targeted genomic manipulation.

Purpose of the Study:

• To develop and characterize a modular CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation.
• To enable simultaneous targeting of multiple genomic loci for complex epigenetic modifications.

Main Methods:

• Utilized orthogonal dCas9 proteins fused to effector domains (DNMT3A, TET1, VPR).
• Implemented a multi-guide RNA (gRNA) system for simultaneous targeting of up to six loci.
• Characterized dCas9 fusions and assessed gene transcription and glycan phenotypes following epigenetic manipulation.

Main Results:

• Demonstrated simultaneous epigenetic editing using DNMT3A-dSpCas9 and TET1-dSaCas9 fusions, altering gene transcription.
• Showed dual epigenetic manipulation of HNF1A and MGAT3 genes changed glycan phenotypes.
• Observed synergistic gene activation with TET1-dSaCas9 and VPR-dSpCas9, persisting for 30 days.
• Modulated dCas9 expression to minimize off-target effects.

Conclusions:

• The developed CRISPR/dCas9 toolbox provides a powerful platform for precise epigenetic editing and gene regulation.
• Simultaneous epigenetic modifications can effectively alter gene transcription and cellular phenotypes.
• Epigenetic mechanisms may play a role in maintaining reactivated gene states.