Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

Area of Science:

• Molecular Biology
• Epigenetics
• Gene Regulation

Background:

• Heritable alteration of gene expression is crucial for biological discovery and therapeutics.
• Existing epigenetic editing tools have limitations in specificity and heritability.

Purpose of the Study:

• To develop a programmable epigenetic memory writer for stable gene silencing.
• To establish rules for heritable gene silencing using genome-wide screens and chromatin analysis.

Main Methods:

• Utilized a dead Cas9 fusion protein (CRISPRoff) to establish DNA methylation and repressive histone modifications.
• Employed transient CRISPRoff expression for gene repression.
• Paired CRISPRoff with genome-wide screens and chromatin mark analysis.

Main Results:

• CRISPRoff successfully initiated specific DNA methylation and gene repression.
• Gene silencing was maintained through cell division and differentiation (stem cells to neurons).
• Identified single guide RNAs (sgRNAs) for silencing most genes, including those lacking CpG islands (CGIs), and demonstrated a wide targeting window beyond CGIs.

Conclusions:

• CRISPRoff provides a versatile platform for heritable gene silencing, expanding the understanding of methylation-based silencing.
• This technology enables diverse applications such as genome-wide screens, multiplexed cell engineering, and enhancer silencing.